U.S. patent application number 15/688718 was filed with the patent office on 2018-03-15 for dendronized polymers for nucleic acid delivery.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Zhibin Guan, Hanxiang Zeng.
Application Number | 20180072849 15/688718 |
Document ID | / |
Family ID | 51569599 |
Filed Date | 2018-03-15 |
United States Patent
Application |
20180072849 |
Kind Code |
A1 |
Guan; Zhibin ; et
al. |
March 15, 2018 |
DENDRONIZED POLYMERS FOR NUCLEIC ACID DELIVERY
Abstract
The disclosure provides for dendronized polymers, and the use of
the polymers as carriers for the intracellular delivery of nucleic
acids.
Inventors: |
Guan; Zhibin; (Irvine,
CA) ; Zeng; Hanxiang; (Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
51569599 |
Appl. No.: |
15/688718 |
Filed: |
August 28, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14221249 |
Mar 20, 2014 |
9745421 |
|
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15688718 |
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61803784 |
Mar 20, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/14 20130101;
C08G 75/14 20130101; C12N 15/111 20130101; C12N 2320/32 20130101;
C12N 15/87 20130101 |
International
Class: |
C08G 75/14 20060101
C08G075/14; C12N 15/87 20060101 C12N015/87; C12N 15/11 20060101
C12N015/11 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with Government support under Grant
No. DMR-0907688, awarded by the National Science Foundation. The
Government has certain rights in the invention.
Claims
1. A dendronzied polymer comprising a highly branched and flexible
architecture that is biocompatible and capable of forming a
polyplex with nucleic acids and releasing the nucleic acids within
a cell.
2. The dendronized polymer of claim 1, comprising the structure of
Formula I: ##STR00028## wherein, n is an integer greater than 50; x
and y are in ratio from 1:99 to 99:1; R.sup.1-R.sup.12 are
independently selected from H, optionally substituted
(C.sub.1-12)-alkyl, optionally substituted
(C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, hydroxyl, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, ether,
amide, amine, imine, azide, cyanate, azo, nitrate, nitrile, nitro,
nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic
acid, sulfonic acid, thiocyanate, thione, thial, phosphine,
phosphonic acid, phosphate, phosphodiester, boronic acid, boronic
ester, borinic acid, and borinic ester; R.sup.13 is an ester;
X.sup.1-X.sup.2 are independently a polyoxyalkylene polymer or an
optionally substituted L-lysine based dendron that is
functionalized on the outer layer by comprising hydrophobic amino
acids and hydrophilic amino acids; and wherein at least one of
X.sup.1-X.sup.2 is an optionally substituted L-lysine based
dendron.
3. The dendronized polymer of claim 2, wherein X.sup.1 has the
structure of: ##STR00029## wherein, m is an integer greater than
100; and R.sup.22-R.sup.36 are independently selected from H,
optionally substituted (C.sub.1-6-alkyl, optionally substituted
(C.sub.1-6)-heteroalkyl, optionally substituted
(C.sub.1-6)-alkenyl, optionally substituted
(C.sub.1-6)-heteroalkenyl, optionally substituted
(C.sub.1-6)-alkynyl, optionally substituted
(C.sub.1-6)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, hydroxyl, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, ether,
amide, amine, imine, azide, cyanate, azo, nitrate, nitrile, nitro,
nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic
acid, sulfonic acid, thiocyanate, thione, thial, phosphine,
phosphonic acid, phosphate, phosphodiester, boronic acid, boronic
ester, borinic acid, borinic ester, methacrylates, acrylates,
maleimides, mesylates, N-hydroxysuccinimide (NHS) esters,
reversible addition-fragmentation chain transfer (RAFT) groups,
tosylates, and biotin.
4. The dendronized polymer of claim 2, wherein the dendronized
polymer comprises a structure of Formula I(a): ##STR00030##
wherein, n is an integer greater than 100; x and y are in ratio
from 1:99 to 99:1; and X.sup.1-X.sup.2 are optionally substituted
L-lysine based dendrons that are functionalized on the outer layer
by comprising hydrophilic-based amino acids and hydrophobic-based
amino acids.
5. The dendronized polymer of claim 2, comprising a structure of
Formula I(b): ##STR00031## wherein, n is an integer greater than
100; m is an integer greater than 100; x and y are in ratio from
5:95 to 95:5; and X.sup.2 is an optionally substituted L-lysine
based dendron that is functionalized on the outer layer by
comprising hydrophilic-based amino acids and hydrophobic-based
amino acids.
6. The dendronized polymer of claim 2, wherein the
hydrophilic-based amino acids are selected from lysine, serine,
histidine, proline, arginine, asparagine, glutamic acid, and
aspartic acid.
7. The dendronized polymer of claim 2, wherein the
hydrophobic-based amino acids are selected from tryptophan,
phenylalanine, tyrosine, leucine, alanine, valine, isoleucine,
methionine, and cysteine.
8. The dendronized polymer of claim 2, wherein the one or more
L-lysine based dendrons comprise hydrophilic amino acids selected
from lysine, histidine, and serine, and hydrophobic amino acids
selected from phenylalanine, tryptophan, and tyrosine, in a molar
ratio of 10:1 to 1:10.
9. The dendronized polymer of claim 8, wherein the molar ratio of
hydrophilic amino acids to the hydrophobic amino acids is 4:1 to
1:4.
10. The dendronized polymer of claim 2, wherein the polyoxyalkylene
polymer is selected from polyethylene glycol (PEG), PEG that has
been functionalized with various functional groups or organic
molecules, PEG diblock copolymers, PEG triblock copolymers,
poly(ethylene glycol-ran-propylene glycol), and poly(ethylene
glycol-ran-propylene glycol) monobutyl ether.
11. The dendronized polymer of claim 2, wherein the polyoxyalkylene
polymer is PEG having a molecular weight between 4,000-10,000.
12. The dendronized polymer of claim 1, wherein the dendronized
polymer comprises a structure of Formula IV: ##STR00032## wherein,
z is an integer greater than one; R.sup.37-R.sup.46 are each
independently selected from H, optionally substituted
(C.sub.1-12)-alkyl, optionally substituted
(C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, hydroxyl, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, ether,
amide, amine, imine, azide, cyanate, azo, nitrate, nitrile, nitro,
nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic
acid, sulfonic acid, thiocyanate, thione, thial, phosphine,
phosphonic acid, phosphate, phosphodiester, boronic acid, boronic
ester, borinic acid, and borinic ester; X.sup.10 is a dendron
comprised of a plurality of linked amino acids that is attached to
the polymer backbone via the sulfide linkage.
13. The dendronized polymer of claim 12, wherein X.sup.6 is a
dendron comprised of a plurality of linked amino acids having the
structure of Formula III: ##STR00033## wherein, v is an integer
greater than one; X.sup.7-X.sup.10 are each independently
hydrophobic or hydrophilic amino acid side groups.
14. The dendronized polymer of claim 1, wherein the dendronized
polymer further comprises a targeting ligand.
15. The dendronized polymer of claim 14, wherein the targeting
ligand is selected from antibodies, aptamers, cholesterol and its
derivatives, folate compounds or folate conjugates, transferrin,
saccharides and cell-penetrating peptides.
16. The dendronized polymer of claim 1, wherein the dendronized
polymer further comprises complexed oligonucleotides or
polynucleotides.
17. The dendronized polymer of claim 16, wherein oligonucleotides
are siRNA.
18. A pharmaceutical composition comprising the dendronized polymer
of claim 17.
19. A method of delivering siRNA to a cell in vitro or in vivo
comprising contacting the cell with the pharmaceutical composition
of claim 18.
20. The method of claim 19, wherein the oligonucleotide induces an
RNAi response in the cell.
21. (canceled)
22. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/221,249, filed Mar. 20, 2014 (now U.S. Pat.
No. 9,745,421), which claims priority under 35 U.S.C. .sctn. 119
from Provisional Application Ser. No. 61/803,784 filed Mar. 20,
2013, the disclosures of which are incorporated herein by
reference.
TECHNICAL FIELD
[0003] The disclosure provides for dendronized polymers, and the
use of the polymers as carriers for the intracellular delivery of
nucleic acids.
BACKGROUND
[0004] RNAi has tremendous potential for therapeutic treatment. The
lack of safe and efficient intracellular delivery of siRNA has
significantly hampered the use of RNAi as a treatment option.
SUMMARY
[0005] The disclosure provides for an innovative biodegradable
peptide-based dendronized polymer ("denpol") architecture that can
be used as a carrier for the intracellular delivery of nucleic
acids. The dendronized polymers disclosed herein combine the
multivalency of dendrimers with the conformational flexibility of
linear polymers for optimal binding of nucleic acids (e.g., siRNA).
By incorporating multi-functional amino acids, the dendronized
polymers of the disclosure were able to overcome various challenges
that impeded the intracellular delivery of nucleic acids. Moreover,
the dendronized polymers of disclosure have versatile structures
that can be tuned both systematically and combinatorially so as to
allow for the optimization of denpols for particular
applications.
[0006] In the Examples provided herein, a focused library was
screened and several denpols were identified that could effectively
deliver siRNA into cells with minimal toxicity in vitro. Moreover,
the denpols of the disclosure had significantly improved
transfection efficiencies over Lipofectamine.TM. (i.e., cationic
lipids) in serum-containing media. In fluorescence intracellular
trafficking studies, it was determined that the amphiphilicity of
the denpols facilitated both cellular uptake and endosomal escape.
For example, it was found that denpols comprising histidine moities
exhibited a buffering capacity that promoted endosomal membrane
rupture, thus enhancing transfection efficacy. The combination of
high delivery efficiency in serum and low cytoxicity demonstrates
that the denpols of the disclosure are effective and safe carriers
for the intracellular delivery of nucleic acids.
[0007] In particular embodiment, the disclosure provides for a
dendronzied polymer comprising a highly branched and flexible
architecture that is biocompatible and capable of forming a
polyplex with nucleic acids and releasing the nucleic acids within
a cell. In a further embodiment, the dendronzied polymer comprises
the structure of Formula I:
##STR00001##
wherein, n is an integer greater than 50; x and y are in ratio from
1:99 to 99:1; R.sup.1-R.sup.12 are independently selected from H,
optionally substituted (C.sub.1-12)-alkyl, optionally substituted
(C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, hydroxyl, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, ether,
amide, amine, imine, azide, cyanate, azo, nitrate, nitrile, nitro,
nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic
acid, sulfonic acid, thiocyanate, thione, thial, phosphine,
phosphonic acid, phosphate, phosphodiester, boronic acid, boronic
ester, borinic acid, and borinic ester; R.sup.13 is an ester;
X.sup.1-X.sup.2 are independently a polyoxyalkylene polymer or an
optionally substituted L-lysine based dendron that is
functionalized on the outer layer by comprising hydrophobic amino
acids and hydrophilic amino acids; and wherein at least one of
X.sup.1-X.sup.2 is an optionally substituted L-lysine based
dendron. In yet further embodiment, X.sup.1 has the structure
of:
##STR00002##
wherein, m is an integer greater than 100; and R.sup.22-R.sup.36
are independently selected from H, optionally substituted
(C.sub.1-6)-alkyl, optionally substituted (C.sub.1-6)-heteroalkyl,
optionally substituted (C.sub.1-6)-alkenyl, optionally substituted
(C.sub.1-6)-heteroalkenyl, optionally substituted
(C.sub.1-6)-alkynyl, optionally substituted
(C.sub.1-6)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, hydroxyl, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, ether,
amide, amine, imine, azide, cyanate, azo, nitrate, nitrile, nitro,
nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic
acid, sulfonic acid, thiocyanate, thione, thial, phosphine,
phosphonic acid, phosphate, phosphodiester, boronic acid, boronic
ester, borinic acid, borinic ester, methacrylates, acrylates,
maleimides, mesylates, N-hydroxysuccinimide (NHS) esters,
reversible addition-fragmentation chain transfer (RAFT) groups,
tosylates, and biotin.
[0008] In a certain embodiment, the disclosure further provides for
a dendronized polymer comprising a structure of Formula I(a):
##STR00003##
wherein, n is an integer greater than 100; x and y are in ratio
from 1:99 to 99:1; and X.sup.1-X.sup.2 are optionally substituted
L-lysine based dendrons that are functionalized on the outer layer
by comprising hydrophilic-based amino acids and hydrophobic-based
amino acids.
[0009] In another embodiment, the disclosure also provides for a
dendronized polymer comprising a structure of Formula I(b):
##STR00004##
wherein, n is an integer greater than 100; m is an integer greater
than 100; x and y are in ratio from 5:95 to 95:5; and X.sup.2 is an
optionally substituted L-lysine based dendron that is
functionalized on the outer layer by comprising hydrophilic-based
amino acids (e.g., lysine, serine, histidine, proline, arginine,
asparagine, glutamic acid, and aspartic acid) and hydrophobic-based
amino acids (e.g., tryptophan, phenylalanine, tyrosine, leucine,
alanine, valine, isoleucine, methionine, and cysteine). In a
further embodiment, the one or more L-lysine based dendrons
comprise hydrophilic amino acids selected from lysine, histidine,
and serine, and hydrophobic amino acids selected from
phenylalanine, tryptophan, and tyrosine, in a molar ratio of 10:1
to 1:10 or a molar ratio of 4:1 to 1:4. In a particular embodiment,
the disclosure for a polyoxyalkylene polymer selected from
polyethylene glycol (PEG), PEG that has been functionalized with
various functional groups or organic molecules, PEG diblock
copolymers, PEG triblock copolymers, poly(ethylene
glycol-ran-propylene glycol), and poly(ethylene
glycol-ran-propylene glycol) monobutyl ether. In yet a further
embodiment, the polyoxyalkylene polymer is PEG having a molecular
weight between 4,000-10,000.
[0010] In a particular embodiment, the disclosure provides for a
dendronized polymer comprising a structure of Formula IV:
##STR00005##
wherein, z is an integer greater than one; R.sup.37-R.sup.46 are
each independently selected from H, optionally substituted
(C.sub.1-12)-alkyl, optionally substituted
(C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, hydroxyl, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, ether,
amide, amine, imine, azide, cyanate, azo, nitrate, nitrile, nitro,
nitroso, thiol, sulfide, disulfide, sulfoxide, sulfone, sulfinic
acid, sulfonic acid, thiocyanate, thione, thial, phosphine,
phosphonic acid, phosphate, phosphodiester, boronic acid, boronic
ester, borinic acid, and borinic ester; and X.sup.6 is a dendron
comprised of a plurality of linked amino acids that is attached to
the polymer backbone via the sulfide linkage. In a further
embodiment, X.sup.6 is a dendron comprised of a plurality of linked
amino acids having the structure of Formula III:
##STR00006##
wherein, v is an integer greater than one; X.sup.7-X.sup.10 are
each independently hydrophobic or hydrophilic amino acid side
groups.
[0011] In a certain embodiment, the disclosure provides for
dendronized polymer that further comprises a targeting ligand.
Examples of targeting ligands include antibodies, aptamers,
cholesterol and its derivatives, folate compounds or folate
conjugates, transferrin, saccharides and cell-penetrating
peptides.
[0012] In a particular embodiment, the disclosure further provides
for a dendronized polymer disclosed herein which further comprises
complexed oligonucleotides or polynucleotides. In a further
embodiment, the complexed oligonucleotides are siRNA.
[0013] In a certain embodiment, the disclosure also provides for
pharmaceutical composition comprising a dendronized polymer/siRNA
polyplex.
[0014] In another embodiment, the disclosure provides for a method
of siRNA to a cell in vitro or in vivo comprising contacting the
cell with the pharmaceutical composition of the disclosure. In yet
a further embodiment, oligonucleotide induces an RNAi response in
the cell.
[0015] In a particular embodiment, the disclosure also provides for
a method of treating a disease or disorder in a subject comprising
administering a pharmaceutical composition disclosed herein.
Examples of diseases or disorders includes diabetes; cancer;
infectious and parasitic diseases; inflammatory diseases;
neurodegenerative diseases; autoimmune diseases; respiratory
diseases; endocrine diseases; eye diseases; intestinal diseases;
cardiovascular diseases; idiopathic diseases; genetic disorders;
growth disorders; congenital disorders; mental or behavioral
disorders; adrenal disorders; thyroid disorders; calcium
homeostasis disorders; pituitary gland disorders; and sex hormone
disorders.
DESCRIPTION OF DRAWINGS
[0016] FIG. 1 illustrates one embodiment of a generalized
architecture of a multifunctional amphiphilic dendronized polymer
disclosed herein.
[0017] FIG. 2 illustrates another embodiment of a generalized
architecture of a multifunctional amphiphilic dendronized polymer
disclosed herein.
[0018] FIG. 3A-B provides a gel permeation chromatography ("GPC")
trace of a denpol backbone before (A) and after (B)
purification.
[0019] FIG. 4 presents a computer generated image of denpol-nucleic
acid polyplex. Multivalent charge and hydrophobic intercalation
lead to stable polyplex formation, but exposure to a reductive
environment leads to the release of the nucleic acid. (Color code:
red=NH2, green=His, blue=Trp).
[0020] FIG. 5 provides a representative .sup.1H NMR peak assignment
(right) of a functionalized denpol (G2 75S-25F (left)) of the
disclosure.
[0021] FIG. 6A-B provides representative .sup.1H NMR peak
assignments for PEGylated denpols: (A) 5% PEG-G1-NH.sub.2, and (B)
5% PEG-G2-75H25W.
[0022] FIG. 7A-G provides images from a gel electrophoresis study
of denpol/siRNA complexation. (A)-(C) Dextran sulfate competition
with different siRNA/denpol polyplexes prepared at N/P 40: (A) G1
75H-25F, (B) G2 75H-25F, (C) G2 75H-25W. (D) siRNA release from
polyplexes prepared at N/P 40 after the treatment of GSH (5 mM) at
r.t. for 30 min ("+": treated with GSH; "-": control). (E)
Transmission electron microscope images of G2 75H-25W/siRNA
polyplex. (F) Atomic force microscope image of G2 75H-25W/siRNA
polyplexes. (G) Dynamic light scattering ("DLS") measurement of G2
75K-25F/siRNA polyplex.
[0023] FIG. 8 provides a DLS measurement of different denpol/siRNA
complexes.
[0024] FIG. 9 provides a DLS measurement of G2-75H25W/siRNA
complexes at different ratios (Note: Due to the presence of excess
free polymer, samples at N/P 80 and 120 are quite polydispersed and
the measurement is less accurate.)
[0025] FIG. 10 provides a dynamic light scattering ("DLS")
measurement of PEGylated denpol/siRNA complexes.
[0026] FIG. 11 presents transmission electron microscopy ("TEM")
images of G2 75H-25W/siRNA complexes at N/P 10.
[0027] FIG. 12A-D characterizes the denpol/siRNA polyplexes of the
disclosure. TEM images of siRNA and G2 75H-25W polyplexes at N/P 10
before (A) and after (B) glutathione treatment. (C) Size
distribution measured by DLS for G2 75H-25W/siRNA polyplexes at N/P
40. (D) MTT assay of selected denpols using the NIH 3T3 cell
line.
[0028] FIG. 13 presents an MTT assay of selected denpols using the
NIH 3T3 cell line.
[0029] FIG. 14 presents in vitro transfection screening results of
select G2 denpols at the optimal N/P ratio by measuring the
reduction of expression of green fluorescent protein ("GFP").
[0030] FIG. 15 presents in vitro transfection screening results of
G1 denpols at an optimal N/P ratio by measuring the reduction of
expression of green fluorescent protein.
[0031] FIG. 16 presents in vitro transfection screening results of
G2 denpols at an optimal N/P ratio by measuring the reduction of
expression of green fluorescent protein.
[0032] FIG. 17 presents MTT cytotoxicity assays of different
PEGylated denpols.
[0033] FIG. 18A-B provides flow cytometry analysis of transfected
NIH 3T3 cells. (A) Transfection summary of selected G2 denpols at
optimal N/P ratio in serum free media. (G2 75H-25W and 75K-25F were
transfected at N/P 80 and the rest at N/P 120). (B) Comparison of
in vitro transfection efficacy between Lipofectamine.RTM. and G2
75H-25W at different serum concentration.
[0034] FIG. 19 presents GFP silencing in 3T3 cells by different
denpols.
[0035] FIG. 20 presents in vitro transfection screening of
PEGylated Denpols in Serum. Different denpols were complexed with
siRNA at optimized N/P ratio (G2-75H25W N/P=120, 5% PEG-G2-75H25W
and 10% PEG-G2-75H25W N/P=400) and transfected to NIH 3T3 cells in
different serum concentrations for 24 hours.
[0036] FIG. 21 provides images from an intracellular fluorescence
trafficking of transfected NIH 3T3 cells. Cells were incubated with
Cy3-labeled siRNA (red) complexed with different denpols for 4
hours. The media was changed back to fresh DMEM with 10% serum.
Fluorescence images were taken at 0 hours, 6 hours or 24 hours
after the transfection. Cell nucleus was counter-stained with DAPI
(blue). Scale bar: 20 .mu.m.
[0037] FIG. 22 provides the same images from an intracellular
fluorescence trafficking of transfected NIH 3T3 cells as in FIG.
21, but at a much higher magnification.
[0038] FIG. 23 provides a schematic of the process used to deliver
siRNA to silence gene expression using the multifunctional
dendronized polypeptide polymers of the disclosure.
DETAILED DESCRIPTION
[0039] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a dendronized polymer" includes a plurality of such dendronized
polymers and reference to "the amino acid" includes reference to
one or more amino acids and equivalents thereof known to those
skilled in the art, and so forth.
[0040] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although many methods and reagents are similar or equivalent to
those described herein, the exemplary methods and materials are
disclosed herein.
[0041] All publications mentioned herein are incorporated by
reference in full for the purpose of describing and disclosing
methodologies that might be used in connection with the description
herein. The publications are provided solely for their disclosure
prior to the filing date of the present application. Nothing herein
is to be construed as an admission that the inventors are not
entitled to antedate such disclosure by virtue of prior disclosure.
Moreover, with respect to any term that is presented in one or more
publications that is similar to, or identical with, a term that has
been expressly defined in this disclosure, the definition of the
term as expressly provided in this disclosure will control in all
respects.
[0042] RNA interference (RNAi) presents tremendous potential as a
new approach in gene therapy. Particularly, small interference RNAs
(siRNAs) has become promising candidates for clinical applications
because of their capability to selectively silence the encoded
protein expression. Since its discovery, a number of siRNA gene
silencing based treatments has reached clinical trials, and the
therapeutic potential of siRNA for a variety of diseases including
cancer, diabetes, and neurodegenerative diseases have been
demonstrated using cell culture as well as animal models. Despite
its potential, therapeutic application of siRNA is greatly hindered
by the lack of safe and effective delivery agents. Both viral and
non-viral delivery agents have been studied extensively in the last
decades. Viral based vectors, although having a higher efficiency
in general, have safety concerns due to their infectious nature and
immunogenicity. On the other hand, synthetic non-viral delivery
agents offer versatile and precise structure control and present as
promising candidates for siRNA delivery. Of these agents, the most
common siRNA delivery agents include cationic lipids, polymers,
dendrimers, peptides and nanoparticles. However, many of these
agents suffer from low efficiencies, high toxicities, and/or are
immunogenic in vivo. Accordingly, very few of these agents have
progressed into clinical trials and none have been approved.
[0043] The disclosure provides for nucleic acid delivery system
comprising dendronized polymers. The dendronized polymers (denpols)
disclosed herein are characterized by the following features: (1)
the denpols are able to form stable polyplexes with nucleic acids,
(2) the denpol/siRNA polyplexes are able to efficiently transport
across cell membrane, (3) the internalized polyplexes must be able
to efficiently escape the endosome before lysosomal degradation,
and (4) the nucleic acid cargo must be able to efficiently
dissociate from the delivery vector in cytosol. The dendronized
polymers of the disclosure were found to be (1) non-toxic and
non-immunogenic, (2) do not negatively interact with blood
components, (3) exhibit desirable pharmacokinectics, and (4) are
able to penetrate tissues to deliver to the target site. The
dendronized polymers disclosed herein are able to provide all these
features and characteristics by (1) having a highly branched
architecture; and (2) having chain flexibility (e.g., see FIG. 1
and FIG. 2). In a particular embodiment, the disclosure also
provides that a dendronized polymer of the disclosure can comprise
amphiphilic moieties which help both cellular uptake and endosomal
escape by enhancing membrane permeability. In a further embodiment,
a dendronized polymer of the disclosure comprises pH responsive
moieties which can facilitate endosomal membrane rupture through a
"proton sponge" effect and/or increasing amphiphilicity at lower
pHs. In yet a further embodiment, the disclosure provides that a
dendronized polymer of the disclosure comprises disulfide bonds
which allow for dissociation of the siRNA in a reducing environment
(i.e., in the cytoplasm of a cell).
[0044] Accordingly, the disclosure provides for innovative
biodegradable dendronized polymers ("denpols") that effectively
deliver nucleic acids (e.g., siRNA) into cells. By contrast to the
teachings of the art, which include studies that indicate that high
generation dendrimers cannot effectively bind and deliver siRNAs
intracellularly, and low generation dendrimers which lack the
multivalency for strong siRNA binding, the dendronized polymers
disclosed herein combine the mulivalency of dendrimers and
conformational flexibility of linear polymers to effectively bind
and deliver siRNA intracellularly. Furthermore, the disclosure
provides methods to make the dendronized polymers disclosed herein
that allow for structural permutation and optimization. In a
particular embodiment, the disclosure provides for a dendronzied
polymer which comprises a highly branched and flexible architecture
that is biocompatible and capable of forming polyplexes with
nucleic acids and releasing the nucleic acids within a cell. In a
further embodiment, a dendronized polymer disclosed herein is fully
composed of natural amino acids so as to ensure biodegradability
and low toxicity.
[0045] It should be understood, however, the disclosure does not
simply provide for dendronized polymers based only on the following
presented structural Formulas, but also includes dendronized
polymers that are comprised of different polymer backbones and
which can contain non-peptide dendrons. Therefore, the dendronized
polymers disclosed herein are not limited to the exemplified
structures presented herein, but include any structure
characterized by the following: a non-toxic and non-immunogenic
polymer that (1) has a highly branched architecture and (2) has
chain flexibility, and which is further capable of forming
polyplexes with nucleic acids and is then able to release these
nucleic acids within a cell. For example, the dendronized polymers
disclosed herein may comprise sugar moieties or a combination of
sugar moieties and amino acid moieties.
[0046] In a particular embodiment, the disclosure provides for a
dendronized polymer comprising the structure of Formula I:
##STR00007##
wherein,
[0047] n is an integer greater than 1;
[0048] x and y are in ratio from 1:99 to 99:1;
[0049] R.sup.1-R.sup.12 are independently selected from the group
comprising H, optionally substituted (C.sub.1-12)-alkyl, optionally
substituted (C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester);
[0050] R.sup.13 is an ester;
[0051] X.sup.1-X.sup.2 are independently a polyoxyalkylene polymer
or an optionally substituted amino acid based dendron that is
functionalized on the outer layer by comprising two different
optionally substituted amino acids; and
[0052] wherein at least one of X.sup.1-X.sup.2 is an optionally
substituted amino acid based dendron.
[0053] In a particular embodiment, n is an integer greater than 10,
50, 100, 500, 1000, 5000, 10000, 15000, or 20000.
[0054] In a further embodiment, the disclosure provides for a
dendronized polymer comprising the structure of Formula I:
##STR00008##
wherein,
[0055] n is an integer greater than 50;
[0056] x and y are in ratio from 1:99 to 99:1;
[0057] R.sup.1-R.sup.12 are independently selected from the group
comprising H, optionally substituted (C.sub.1-12)-alkyl, optionally
substituted (C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester);
[0058] R.sup.13 is an ester;
[0059] X.sup.1-X.sup.2 are independently a polyoxyalkylene polymer
or an optionally substituted L-lysine based dendron that is
functionalized on the outer layer by comprising hydrophobic amino
acids and hydrophilic amino acids; and
[0060] wherein at least one of X.sup.1-X.sup.2 is an optionally
substituted L-lysine based dendron.
[0061] Examples of hydrophilic-based amino acids include, but are
not limited to, lysine, serine, histidine, proline, arginine,
asparagine, glutamic acid, and aspartic acid.
[0062] Examples of hydrophobic-based amino acids include, but are
not limited to, tryptophan, phenylalanine, tyrosine, leucine,
alanine, valine, isoleucine, methionine, and cysteine.
[0063] Examples of polyoxyalkylene polymers, include but are not
limited to: polyethylene glycol (PEG); PEG which has been
functionalized with various functional groups or organic molecules,
including: halides, acetylenes, amines, azides, hydroxyls, thiols,
methacrylates, acrylates, carboxylic acids, maleimides, mesylates,
NHS esters, RAFT groups, tosylates, biotin or any combination of
the foregoing; PEG diblock copolymers, including PEG-PLA, PEG-PLGA,
PEG-PCL, PEG-PE, and PEG-PS; PEG triblock copolymers, including
PEG-PPG-PEG, PPG-PEG-PPG, PLA-PEG-PLA, PLGA-PEG-PLGA, and
PLCL-PEG-PLCL; poly(ethylene glycol-ran-propylene glycol); and
poly(ethylene glycol-ran-propylene glycol) monobutyl ether. Most if
not all of these polyoxyalkylene polymers are commercially
available from various vendors, such as Sigma-Aldrich (St. Louis,
Mo.). Furthermore, a person of ordinary skill in the art would
recognize that these polymers can readily be incorporated into
synthesis methods presented herein (e.g., SCHEME 7) to produce a
denpol of the disclosure (e.g., a polyoxyalkylene-denpol).
Additionally, these polyoxyalkylene polymers come in various
molecular weights and it is fully contemplated by this disclosure
that any molecular size polyoxyalkylene polymer can be used to make
a dendronized polymer of the disclosure. For example, PEG having an
average molecular weight of about 200, about 300, about 400, about
600, about 1000, about 1450, about 1500, about 2000, about 3000,
about 3350, about 4000, about 4600, about 5000, about 6000, about
8000, about 10000, about 12,000, about 20,000, about 35,000, or in
a range between any two of the foregoing, can all be used in the
synthesis of a denpol of the disclosure.
[0064] In a one embodiment, X.sup.1-X.sup.2 comprise optionally
substituted L-lysine based dendron that is functionalized on the
outer layer by comprising a ratio of optionally substituted
hydrophilic amino acids and optionally substituted hydrophobic
amino acids. In a particular embodiment, a dendronized polymer
disclosed herein is functionalized with a ratio of
hydrophilic-based amino acids to hydrophobic-based amino acids in
the range of 20:1 to 1:20; 15:1 to 15:1; 10:1 to 1:10; 9:1 to 1:9,
8:1 to 1:8; 7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5; 4:1 to 1:4; 3:1 to
1:3; 3:2 to 2:3; 2:1 to 1:2; 1.5:1 to 1:1.5; or about 1:1. In a
further embodiment, a dendronized polymer disclosed herein is
functionalized with a ratio of a first amino acid selected from
lysine, histidine, and serine, to a second amino acid selected from
phenylalanine, tryptophan, tyrosine, and leucine, wherein the ratio
to the first amino acid to the second amino acid is in the range of
20:1 to 1:20; 15:1 to 15:1; 10:1 to 1:10; 9:1 to 1:9, 8:1 to 1:8;
7:1 to 1:7, 6:1 to 1:6, 5:1 to 1:5; 4:1 to 1:4; 3:1 to 1:3; 3:2 to
2:3; 2:1 to 1:2; 1.5:1 to 1:1.5; or about 1:1. In one embodiment, a
dendronized polymer disclosed herein is functionalized with a ratio
of histidine to tryptophan in the range of 5:1 to 1:5; 4:1 to 1:4;
3:1 to 1:3; 3:2 to 2:3; 2:1 to 1:2; 1.5:1 to 1:1.5; or about
1:1.
[0065] In a further embodiment, the disclosure provides for a
L-lysine based dendron comprising the structure of Formula II:
##STR00009##
wherein,
[0066] R.sup.14-R.sup.21 are independently selected from the group
comprising H, optionally substituted (C.sub.1-6)-alkyl, optionally
substituted (C.sub.1-6)-heteroalkyl, optionally substituted
(C.sub.1-6)-alkenyl, optionally substituted
(C.sub.1-6)-heteroalkenyl, optionally substituted
(C.sub.1-6)-alkynyl, optionally substituted
(C.sub.1-6)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester);
[0067] X.sup.3-X.sup.4 are independently selected from L.sup.1,
optionally substituted hydrophobic amino acids, and optionally
substituted hydrophilic amino acids.
[0068] In a particular embodiment, the disclosure provides for a
dendronized polymer comprising a polymer backbone Formula I:
##STR00010##
wherein,
[0069] n is an integer greater than 100;
[0070] x and y are in ratio from 1:99 to 99:1;
[0071] R.sup.1-R.sup.12 are individually selected from the group
comprising H, optionally substituted (C.sub.1-12)-alkyl, optionally
substituted (C.sub.1-12)-heteroalkyl, optionally substituted
(C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester);
[0072] R.sup.13 is an ester;
[0073] X.sup.1 has the structure of:
##STR00011##
[0074] wherein m is an integer greater than 100, and
R.sup.22-R.sup.36 are independently selected from H, optionally
substituted (C.sub.1-6)-alkyl, optionally substituted
(C.sub.1-6)-heteroalkyl, optionally substituted
(C.sub.1-6)-alkenyl, optionally substituted
(C.sub.1-6)-heteroalkenyl, optionally substituted
(C.sub.1-6)-alkynyl, optionally substituted
(C.sub.1-6)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester), and R.sup.32 is selected from H, optionally substituted
(C.sub.1-6)-alkyl, optionally substituted (C.sub.1-6)-heteroalkyl,
optionally substituted (C.sub.1-6)-alkenyl, optionally substituted
(C.sub.1-6)-heteroalkenyl, optionally substituted
(C.sub.1-6)-alkynyl, optionally substituted
(C.sub.1-6)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester), methacrylates, acrylates, maleimides, mesylates,
N-hydroxysuccinimide (NHS) esters, reversible
addition-fragmentation chain transfer (RAFT) groups, tosylates, and
biotin; and
[0075] X.sup.2 is an optionally substituted L-lysine based dendron
that is functionalized on the outer layer by comprising
hydrophilic-based amino acids and hydrophobic-based amino
acids.
[0076] In a particular embodiment, the disclosure provides for a
dendronized polymer comprising a structure of Formula I(a):
##STR00012##
wherein,
[0077] n is an integer greater than 100;
[0078] x and y are in ratio from 1:99 to 99:1; and
[0079] X.sup.1-X.sup.2 are optionally substituted L-lysine based
dendrons that are functionalized on the outer layer by comprising
hydrophilic-based amino acids and hydrophobic-based amino
acids.
[0080] In another embodiment, the disclosure provides for a
dendronized polymer comprising a structure of Formula I(b):
##STR00013##
wherein,
[0081] n is an integer greater than 100;
[0082] m is an integer greater than 100;
[0083] x and y are in ratio from 5:95 to 95:5; and
[0084] X.sup.2 is an optionally substituted L-lysine based dendron
that is functionalized on the outer layer by comprising
hydrophilic-based amino acids and hydrophobic-based amino
acids.
[0085] In a particular embodiment, the disclosure provides for a
dendronized polymer comprising a structure of Formula III:
##STR00014##
wherein,
[0086] z is an integer greater than one;
[0087] R.sup.37-R.sup.46 are each independently selected from the
group comprising H, optionally substituted (C.sub.1-12)-alkyl,
optionally substituted (C.sub.1-12)-heteroalkyl, optionally
substituted (C.sub.1-12)-alkenyl, optionally substituted
(C.sub.1-12)-heteroalkenyl, optionally substituted
(C.sub.1-12)-alkynyl, optionally substituted
(C.sub.1-12)-heteroalkynyl, optionally substituted cycloalkyl,
optionally substituted cycloalkenyl, halide, optionally substituted
oxygen containing functional group (e.g., alcohol, ketone,
aldehyde, acyl halide, carbonate, carboxylic acid, ester, and
ether), optionally substituted nitrogen containing functional group
(e.g., amide, amine, imine, azide, cyanate, azo, nitrate, nitrile,
nitro, and nitroso), optionally substituted sulfur containing
functional group (e.g., thiol, sulfide, disulfide, sulfoxide,
sulfone, sulfinic acid, sulfonic acid, thiocyanate, thione, and
thial), optionally substituted phosphorous containing functional
group (e.g., phosphine, phosphonic acid, phosphate,
phosphodiester), optionally substituted boron containing functional
group (e.g., boronic acid, boronic ester, borinic acid, and borinic
ester);
[0088] X.sup.6 is a dendron comprised of a plurality of linked
amino acids that is attached to the polymer backbone via a sulfide
linkage.
[0089] In a certain embodiment, X.sup.6 is a dendron comprised of a
plurality of linked amino acids having the structure of Formula
III:
##STR00015##
wherein,
[0090] v is an integer greater than one;
[0091] X.sup.7-X.sup.10 are each independently hydrophobic or
hydrophilic amino acid side groups.
[0092] The disclosure further provides that a "graft-from" approach
can be used to produce the dendronized polymers of the disclosure.
For example, dendronized polymers comprising the structure of
Formula I can be made by following the generalized "graft from"
method of SCHEME I:
##STR00016## ##STR00017##
[0093] In a particular embodiment, a "graft-from" approach can be
used to construct denpols of the disclosure (SCHEME 1). The
backbone of denpol is prepared by simple in situ peptide coupling
polymerization between a dicysteine monomer 1 and a lysine monomer
2 (polymer M.sub.n.about.15KD, PDI.about.1.8 by GPC). The disulfide
linkages on the polymer backbone are introduced to be biodegradable
under a reducing environment in the cytoplasm so as to facilitate
nucleic acid decomplexation. After Boc deprotection, lysine-based
dendrons were grown from the polymer backbone 4 generation by
generation through solution phase peptide coupling. Finally,
hydrophilic and hydrophobic amino acids at different molar ratios
were coupled to the outer layer of the scaffold to introduce
different functionalities 5. The chemical structures of the final
denpols can then be characterized by .sup.1H NMR analysis.
Following this "graft-from" protocol, a small focused library of
amphiphilic denpols was quickly generated. Lysine and serine were
chosen for the hydrophilic moiety with either charged or neutral
side chain, and histidine was chosen for its good buffering
capacity, which could help the endosomal escape by the "proton
sponge" mechanism. Amino acids carrying aromatic or aliphatic
hydrophobic group were incorporated into the denpol with different
ratio. Throughout this disclosure, single letter amino acid codes
will be used for naming. For example, G2 75H-25W represents a
denpol with gen-2 dendrons composed of 75 mol % histidine (H) and
25 mol % tryptophan (W) residues on dendrons.
[0094] In an alternate embodiment, the disclosure provides a
"combinatorial" approach can be used to produce the dendronized
polymers of the disclosure. For example, dendronized polymers
comprising the structure of Formula III can be made by following
the generalized "combinatorial" method of SCHEME 2:
##STR00018##
[0095] In a particular embodiment, oligolysine can be used to
construct the polymer backbone. Oligolysine can be facilely made by
SPPS. Following procedure of Kantchev et al. (Org Biomol Chem
6(8):1377-85 (2008)), a library of dendrons with controlled size
and functionalities are synthesized by automated SPPS (SCHEME 2).
Briefly, Rink Amide Resin is first coupled with cysteine, which is
then used as an anchoring group for grafting to the polymer
backbone through a disulfide linkage. Lysine dendron is grown step
by step until reaching the desired generation. Finally, the out
layer is functionalized with a combination of hydrophilic (R.sub.1)
and hydrophobic (R.sub.2) amino acids. Each of the outer layer
amino acid residues carries one positive charge from the
.alpha.-amino group, providing the base level of cationic charge
density for the denpols. Depending on the coupling protocol, the
spatial placement of different functional groups on the dendrons
can be precisely controlled. After acid cleavage, each individual
dendron is purified and characterized. SPPS is ideally suited
because it provides expedient access to a large library of
dendrons. Once promising dendrons are identified in later studies,
standard solution phase synthesis can be easily adapted for making
larger quantities of the identified dendrons for further
studies.
[0096] In another embodiment, the disclosure provides a "graft-to"
approach can be used to produce the dendronized polymers of the
disclosure. For example, dendronized polymers comprising the
structure of Formula IV can be made by following the generalized
"graft-to" method of SCHEME 3:
##STR00019##
[0097] In a certain embodiment, a saccharide-peptide hybrid polymer
(SPHP, 6) can be used to form a denpol of the disclosure. This
polymer features facile synthesis, biodegradability, versatile
functionalization and is nontoxic and non-immunogenic. The dendrons
will be conjugated to the SPHP polymer backbone via disulfide
bonds, which can be cleaved by cytosolic glutathione (GSH) to
facilitate decomplexation of siRNA in cytosol. For this purpose,
cysteine will be first coupled to the polymer backbone and then
activated by 2,2'-dithioldipyridine. With this universal backbone
8, cysteine-terminated dendrons (G1-G3) can be conveniently grafted
to polymer backbone via a thiol exchange reaction (SCHEME 3).
[0098] In a particular embodiment, a denpol disclosed herein
comprises both hydrophilic and hydrophobic amino acids. In a
further embodiment the denpol of the disclosure comprises one or
more hydrophilic amino acids based on (1) moieties that have a
positive charge that can facilitate binding with siRNAs (e.g.,
lysine (K) and arginine (R)); (2) moieties which can form hydrogen
bonds (e.g., serine (S) and asparagine (N)); moieties that have
good buffering capacity (e.g., histidine (H)). In another
embodiment, the hydrophobic amino acids are hydrophobic aromatic
amino acids (e.g., tryptophan (W), phenylalanine (F), and tyrosine
(Y)). The indole ring of W can intercalate into double stranded
siRNA and therefore increase binding strength. In addition, indole
(W) and phenol (Y) groups are cytoprotective antioxidant that may
help subside the oxidative immune response and improve cell
viability after transfection. Use aforementioned methods, a binary
library of G1-G3 dendrons can be synthesized which comprise
hydrophobic amino acid residues and hydrophilic amino acid residues
in a molar ratio of hydrophilic/hydrophobic ranging from 90:10,
75:25, or 60:40.
[0099] In another embodiment, the disclosure provides for
systematic tuning of the spatial arrangement of the functional
groups. The spatial placement of ligands can greatly affect the
receptor binding and downstream biological response. A comparative
study of the following three types of spatial arrangements (SCHEME
4A-C) can be performed as follows to optimize the spatial
arrangement of the functional groups to meet a specific
application. First, a random hybrid dendron is prepared by adding a
mixture of two different amino acids at the end of dendron
synthesis (SCHEME 2) to distribute the functional groups randomly
on the outer layer (SCHEME 4A). A uniform hybrid dendron is then
prepared by using Fmoc-Lys(Cbz)-OH in dendron preparation.
Selective deprotection and subsequent coupling allows for the
precise placement of different functional group at each position on
the outer layer (SCHEME 4B). Two mono-functional dendrons are also
prepared, which will be co-grafted onto the denpol backbone at the
desired ratio (SCHEME 4C). All three types of denpols are prepared
to have the same composition, and their biological properties are
then compared in subsequent studies. Second, in conjugating
dendrons onto polymer backbone, the space between dendrons can be
controlled by the grafting density, which is modulated by the molar
ratio of dendron to polymer backbone. The remaining functional
sites are capped by a concurrent reaction with 2-mercaptoethanol
(HSCH.sub.2CH.sub.2OH).
##STR00020##
[0100] In a further embodiment, a dendronized polymer disclosed
herein further comprises targeting ligands. Examples of targeting
ligands, include but are not limited to, antibodies, aptamers,
cholesterol and its derivatives, folate compounds or folate
conjugates, transferrin, saccharides (e.g., mono-, di-,
oligo-saccharides), and cell-penetrating peptides. These targeting
ligands can be conjugated to the dendronized polymers by using the
techniques presented in Shu et al. (Annual Review of Physical
Chemistry 64:631-657 (2013)), Gauthier et al. (Chem. Commun
23:2591-2611 (2008)), Menzel (Advances in Polymer Science 253:1-36
(2013)), Mero et al. (Methods Mol Biol. 751:95-129 (2011)), Roberts
et al. (Advanced Drug Delivery Reviews 54:459-476 (2002)), Steenis
et al. (Journal of Controlled Release 87:167-176 (2003)), which are
incorporated herein in-full, including the references cited
therein.
[0101] In another embodiment, the dendronized polymers disclosed
herein further comprise an oligonucleotide (e.g., siRNA) or a
polynucleotide.
[0102] In a particular embodiment, the disclosure provides methods
for delivering an oligonucleotide or polynucleotide to a cell in
vitro or in vivo comprising contacting the cell with a
pharmaceutical composition disclosed herein or a dendronized
polymer of the disclosure. In a further embodiment, the disclosure
provides methods for inducing an RNAi response in a cell by
delivering a siRNA into a cell by using a dendronized polymer
disclosed herein.
[0103] Extracellularly, siRNAs are highly susceptible to
degradation by enzymes found in serum and tissues. The half-life of
naked siRNAs in serum ranges from several minutes to an hour. The
large size and negative charge of naked siRNAs thwarts their
diffusion across the plasma membrane and prevents intracellular
accumulation. siRNA delivery strategies that take advantage of
endocytosis also must provide for endosomal escape. In the
experiments presented herein, the dendronized polymers of the
disclosure can form complexes with siRNA, are stable in serum,
allow for siRNA diffusion across the plasma membrane, and provide
for endosomal escape. Accordingly, the dendronized polymers
disclosed herein are particularly suited for delivering siRNAs to
cells.
[0104] As used herein, a nucleic acid domain, used interchangeably
with oligonucleotide or polynucleotide domain can be any
oligonucleotide or polynucleotide (e.g., a ribozyme, antisense
molecule, siRNA, dsRNA, polynucleotide, oligonucleotide and the
like). Oligonucleotides or polynucleotides generally contain
phosphodiester bonds, although in some cases, nucleic acid analogs
are included that may have alternate backbones, comprising, e.g.,
phosphoramidate, phosphorothioate, phosphorodithioate, or
O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides
and Analogues: A Practical Approach, Oxford University Press); and
peptide nucleic acid backbones and linkages. Other analog nucleic
acids include those with positive backbones; non-ionic backbones,
and non-ribose backbones, including those described in U.S. Pat.
Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium
Series 580, Carbohydrate Modifications in Antisense Research,
Sanghui & Cook, eds. Nucleic acids containing one or more
carbocyclic sugars are also included within one definition of
nucleic acids. Modifications of the ribose-phosphate backbone may
be done for a variety of reasons, e.g. to increase the stability
and half-life of such molecules in physiological environments.
Oligonucleotides, as used this disclosure, therefore encompass
siRNAs which have been chemically modified to prolong the siRNA
half-life in serum and increased cellular uptake. Examples of such
modifications, including modifying the sugar moiety by
incorporating a 2'-fluoro, 2'-omethyl, 2'-halogen, 2'-amine, or
2-'deoxy, or by bridging the sugar's 2' and 4' positions with a
--O--CH.sub.2 linker (i.e., a `locked nucleic acid`); by modifying
the internucleotide phosphate linkage in siRNA by replacing the
phosphodiester linkage with phosphothioate or boranophosphate; by
modifying the siRNA nucleobases by replacing uridine bases with
4-thiouridine, 5-bromouridine, 5-iodouridine, N-3-Me-uridine or
2,6-diaminopurine residues, or by replacing seed region nucleotides
2-8 (from the 5'end of the guide strand) of siRNA with DNA
nucleotides. Mixtures of naturally occurring nucleic acids and
analogs are encompassed by the term oligonucleotide and
polynucleotide; alternatively, mixtures of different nucleic acid
analogs, and mixtures of naturally occurring nucleic acids and
analogs can be made. Furthermore, hybrids of DNA and RNA can be
used. dsDNA, ssDNA, dsRNA, siRNA are encompassed by the term
oligonucleotide and polynucleotide. Additionally, the term
oligonucleotides and polynucleotides, as used herein, includes
modifications of siRNA termini, including tagging the ends of
siRNAs with moieties such as cholesterol, folate, various peptides,
and aptamers; fluorescent molecules; 3'-biotin; and 3'-ends with
dinucleotide overhangs that mimic Dicer cleavage products.
[0105] A polynucleotide refers to a polymeric compound made up of
any number of covalently bonded nucleotide monomers, including
nucleic acid molecules such as DNA and RNAmolecules, including
single- double- and triple-stranded such molecules, and is
expressly intended to embrace that group of polynucleotides
commonly referred to as "oligonucleotides", which are typically
distinguished as having a relatively small number (no more than
about 30, e.g., about 5-10, 10-20, and 20-30) of nucleotide
bases.
[0106] As used herein, the term "siRNA" is an abbreviation for
"short interfering RNA", also sometimes known as "small interfering
RNA" or "silencing RNA", and refers to a class of nucleotide-long
double-stranded ribonucleic acid molecules that in eukaryotes are
involved in the RNA interference (RNAi) pathway that results in
post-transcriptional, sequence-specific gene silencing.
[0107] The term "dsRNA" is an abbreviation for "double-stranded
RNA" and as used herein refers to a ribonucleic acid molecule
having two complementary RNA strands.
[0108] As described above, the nucleic acid may be DNA, both
genomic and cDNA, RNA or a hybrid, where the nucleic acid may
contain combinations of deoxyribo- and ribo-nucleotides, and
combinations of bases, including uracil, adenine, thymine,
cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,
isoguanine, etc. As used herein, the term "nucleoside" includes
nucleotides and nucleoside and nucleotide analogs, and modified
nucleosides such as amino modified nucleosides. In addition,
"nucleoside" includes non-naturally occurring analog structures.
Thus, e.g. the individual units of a peptide nucleic acid, each
containing a base, are referred to herein as a nucleoside.
[0109] The nucleic acid domain of a nucleic acid construct
described herein is not limited by any particular sequence. Any
number of oligonucleotide or polynucleotides useful for
diagnostics, therapeutics and research can be used in the methods
and compositions of the disclosure.
[0110] The practice of phosphoramidite chemistry to prepare
oligonucleotides is known from the published work of M. Caruthers
and S. Beaucage and others. U.S. Pat. Nos. 4,458,066, 4,500,707,
5,132,418, 4,415,732, 4,668,777, 4,973,679, 5,278,302, 5,153,319,
5,218,103, 5,268,464, 5,000,307, 5,319,079, 4,659,774, 4,672,110,
4,517,338, 4,725,677 and Re. 34,069, each of which is herein
incorporated by reference, describe methods of oligonucleotide
synthesis. Additionally, the practice of phosphoramidite chemistry
has been systematically reviewed by Beaucage and Iyer (Tetrahedron
48:2223-2311 (1942)) and (Tetrahedron 49:6123-6194 (1993)), or
references referred to therein, all of which are herein
incorporated by reference.
[0111] Nucleic acid synthesizers are commercially available and
their use is generally understood by persons of ordinary skill in
the art as being effective in generating nearly any oligonucleotide
of reasonable length which may be desired.
[0112] In practicing phosphoramidite chemistry useful 5'OH sugar
blocking groups are trityl, momomethoxytrityl, dimethoxytrityl and
trimethoxytrityl, especially dimethoxytrityl (DMTr). In practicing
phosphoramidite chemistry useful phosphite activating groups, i.e.,
NR.sub.2, are dialkyl substituted nitrogen groups and nitrogen
heterocycles. One approach includes the use of the
di-isopropylamino activating group.
[0113] Oligonucleotides can be synthesized by a Mermade-6 solid
phase automated oligonucleotide synthesizer or any commonly
available automated oligonucleotide synthesizer. Triester,
phosphoramidite, or hydrogen phosphonate coupling chemistries
described in, for example, M. Caruthers, Oligonucleotides:
Antisense Inhibitors of Gene Expression., pp. 7-24, J. S. Cohen,
ed. (CRC Press, Inc. Boca Raton, Fla., 1989) or Oligonucleotide
synthesis, a practical approach, Ed. M. J. Gait, IRL Press, 1984;
"Oligonucleotides and Analogues, A Practical Approach", Ed. F.
Eckstein, IRL Press, 1991, are employed by these synthesizers to
provide the desired oligonucleotides. The Beaucage reagent, as
described in, for example, Journal of American Chemical Society
112:1253-1255 (1990), or elemental sulfur, as described in Beaucage
et al., (Tetrahedron Letters 22:1859-1862 (1981)), is used with
phosphoramidite or hydrogen phosphonate chemistries to provide
substituted phosphorothioate oligonucleotides. For example, the
reagents comprising the protecting groups recited herein can be
used in numerous applications where protection is desired. Such
applications include, but are not limited to, both solid phase and
solution phase, oligo-synthesis, polynucleotide synthesis and the
like. The use of nucleoside and nucleotide analogs is also
contemplated by this disclosure to provide oligonucleotide or
oligonucleoside analogs bearing the protecting groups disclosed
herein. Thus the terms nucleoside, nucleotide, deoxynucleoside and
deoxynucleotide generally include analogs such as those described
herein. These analogs are those molecules having some structural
features in common with a naturally occurring nucleoside or
nucleotide such that when incorporated into an oligonucleotide or
oligonucleoside sequence, they allow hybridization with a naturally
occurring oligonucleotide sequence in solution. Typically, these
analogs are derived from naturally occurring nucleosides and
nucleotides by replacing and/or modifying the base, the ribose or
the phosphodiester moiety. The changes can be tailor made to
stabilize or destabilize hybrid formation or enhance the
specificity of hybridization with a complementary nucleic acid
sequence as desired.
[0114] For instance, structural groups are optionally added to the
ribose or base of a nucleoside for incorporation into an
oligonucleotide, such as a methyl, propyl or allyl group at the
2'-0 position on the ribose, or a fluoro group which substitutes
for the 2'-O group, or a bromo group on the ribonucleoside base.
For use with phosphoramidite chemistry, various amidite reagents
are commercially available, including 2'-deoxy amidites,
2'-O-methyl amidites and 2'-O-hydroxyl amidites. Any other means
for such synthesis may also be employed. The actual synthesis of
the oligonucleotides is well within the talents of those skilled in
the art. It is also well known to use similar techniques to prepare
other oligonucleotides such as the phosphorothioates, methyl
phosphonates and alkylated derivatives. It is also well known to
use similar techniques and commercially available modified amidites
and controlled-pore glass (CPG) products such as biotin, Cy3,
fluorescein, acridine or psoralen-modified amidites and/or CPG
(available from Glen Research, Sterling Va.) to synthesize
fluorescently labeled, biotinylated or other conjugated
oligonucleotides.
[0115] In a certain embodiment, the disclosure provides for a
pharmaceutical composition which comprises the dendronized polymers
of the disclosure. Moreover, the pharmaceutical composition can be
formulated into a form suitable for administration to a subject
including the use of carriers, excipients, additives or
auxiliaries. Frequently used carriers or auxiliaries include
magnesium carbonate, titanium dioxide, lactose, mannitol and other
sugars, talc, milk protein, gelatin, starch, vitamins, cellulose
and its derivatives, animal and vegetable oils, polyethylene
glycols and solvents, such as sterile water, alcohols, glycerol,
and polyhydric alcohols. Intravenous vehicles include fluid and
nutrient replenishers. Preservatives include antimicrobial,
anti-oxidants, chelating agents, and inert gases. Other
pharmaceutically acceptable carriers include aqueous solutions,
non-toxic excipients, including salts, preservatives, buffers and
the like, as described, for instance, in Remington's Pharmaceutical
Sciences, 15th ed., Easton: Mack Publishing Co., 1405-1412,
1461-1487 (1975), and The National Formulary XIV., 14th ed.,
Washington: American Pharmaceutical Association (1975), the
contents of which are hereby incorporated by reference. The pH and
exact concentration of the various components of the pharmaceutical
composition are adjusted according to routine skills in the art.
See Goodman and Gilman's, The Pharmacological Basis for
Therapeutics (7th ed.).
[0116] The pharmaceutical compositions according to the disclosure
may be administered at a therapeutically effective amount either
locally or systemically. As used herein, "administering a
therapeutically effective amount" is intended to include methods of
giving or applying a pharmaceutical composition of the disclosure
to a subject that allow the composition to perform its intended
therapeutic function. The therapeutically effective amounts will
vary according to factors, such as the degree of infection in a
subject, the age, sex, and weight of the individual. Dosage regime
can be adjusted to provide the optimum therapeutic response. For
example, several divided doses can be administered daily or the
dose can be proportionally reduced as indicated by the exigencies
of the therapeutic situation.
[0117] The pharmaceutical composition can be administered in a
convenient manner, such as by injection (e.g., subcutaneous,
intravenous, and the like), oral administration, inhalation,
transdermal application, or rectal administration. Depending on the
route of administration, the pharmaceutical composition can be
coated with a material to protect the pharmaceutical composition
from the action of enzymes, acids, and other natural conditions
that may inactivate the pharmaceutical composition. The
pharmaceutical composition can also be administered parenterally or
intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof, and in oils.
Under ordinary conditions of storage and use, these preparations
may contain a preservative to prevent the growth of
microorganisms.
[0118] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. The composition
will typically be sterile and fluid to the extent that easy
syringability exists. Typically the composition will be stable
under the conditions of manufacture and storage and preserved
against the contaminating action of microorganisms, such as
bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size, in the case of dispersion, and by the use
of surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, isotonic agents, for
example, sugars, polyalcohols, such as mannitol, sorbitol, or
sodium chloride are used in the composition. Prolonged absorption
of the injectable compositions can be brought about by including in
the composition an agent that delays absorption, for example,
aluminum monostearate and gelatin.
[0119] Sterile injectable solutions can be prepared by
incorporating the pharmaceutical composition in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
pharmaceutical composition into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from
those enumerated above.
[0120] The pharmaceutical composition can be orally administered,
for example, with an inert diluent or an assimilable edible
carrier. The pharmaceutical composition and other ingredients can
also be enclosed in a hard or soft-shell gelatin capsule,
compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the
pharmaceutical composition can be incorporated with excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% by weight
of active compound. The percentage of the compositions and
preparations can, of course, be varied and can conveniently be
between about 5% to about 80% of the weight of the unit.
[0121] The tablets, troches, pills, capsules, and the like can also
contain the following: a binder, such as gum gragacanth, acacia,
corn starch, or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid, and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin, or a
flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring. When the dosage unit form is a capsule, it can contain,
in addition to materials of the above type, a liquid carrier.
Various other materials can be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules can be coated with shellac, sugar, or both. A
syrup or elixir can contain the agent, sucrose as a sweetening
agent, methyl and propylparabens as preservatives, a dye, and
flavoring, such as cherry or orange flavor. Of course, any material
used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic in the amounts employed. In
addition, the pharmaceutical composition can be incorporated into
sustained-release preparations and formulations.
[0122] Thus, a "pharmaceutically acceptable carrier" is intended to
include solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the pharmaceutical
composition, use thereof in the therapeutic compositions and
methods of treatment is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0123] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form" as used herein, refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
pharmaceutical composition is calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier. The specification for the dosage unit forms of the
disclosure are related to the characteristics of the pharmaceutical
composition and the particular therapeutic effect to be
achieve.
[0124] The principal pharmaceutical composition is compounded for
convenient and effective administration in effective amounts with a
suitable pharmaceutically acceptable carrier in an acceptable
dosage unit. In the case of compositions containing supplementary
active ingredients, the dosages are determined by reference to the
usual dose and manner of administration of the said
ingredients.
[0125] For use in the therapeutic applications described herein,
kits and articles of manufacture are also described herein. Such
kits can comprise a carrier, package, or container that is
compartmentalized to receive one or more containers such as vials,
tubes, and the like, each of the container(s) comprising one of the
separate elements to be used in a method described herein. Suitable
containers include, for example, bottles, vials, syringes, and test
tubes. The containers can be formed from a variety of materials
such as glass or plastic.
[0126] For example, the container(s) can comprise one or more
denpols described herein, optionally in a composition or in
combination with another agent (e.g., siRNAs) as disclosed herein.
The container(s) optionally have a sterile access port (for example
the container can be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). Such kits
optionally comprise an identifying description or label or
instructions relating to its use in the methods described
herein.
[0127] A kit will typically comprise one or more additional
containers, each with one or more of various materials (such as
reagents, optionally in concentrated form, and/or devices)
desirable from a commercial and user standpoint for use of a
compound described herein. Non-limiting examples of such materials
include, but are not limited to, buffers, diluents, filters,
needles, syringes; carrier, package, container, vial and/or tube
labels listing contents and/or instructions for use, and package
inserts with instructions for use. A set of instructions will also
typically be included.
[0128] A label can be on or associated with the container. A label
can be on a container when letters, numbers or other characters
forming the label are attached, molded or etched into the container
itself, a label can be associated with a container when it is
present within a receptacle or carrier that also holds the
container, e.g., as a package insert. A label can be used to
indicate that the contents are to be used for a specific
therapeutic application. The label can also indicate directions for
use of the contents, such as in the methods described herein. These
other therapeutic agents may be used, for example, in the amounts
indicated in the Physicians' Desk Reference (PDR) or as otherwise
determined by one of ordinary skill in the art.
[0129] The following examples are intended to illustrate but not
limit the disclosure. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
Examples
[0130] Materials.
[0131] Unless otherwise noticed, all reagents were used as received
from commercial suppliers without further purification. Protected
amino acids were purchased from Advanced ChemTech (Loiusville, Ky.)
and Aroz Technologies, LLC. (Cincinnati, Ohio). Coupling reagents
were purchased from GL Biochem Ltd. (Shanghai, China). Branched
polyethyleneimine (PEI, 25 kDa) was purchased from Sigma-Aldrich
(St. Louis, Mo.). Sodium Dextran Sulfate (25 kDa) was purchased
from TCI America (Portland, Oreg.) and was used as received.
GelRed.TM. siRNA stain was purchased from VWR (Radnor, Pa.).
Silencer anti-GFP siRNA, Silencer Select negative control siRNA,
Silencer Cy.TM.-3 labeled Negative Control siRNA and
Lipofectamine.RTM. RNAiMAX were purchased from Invitrogen
(Carlsbad, Calif.). All reactions were performed in HPLC grade
unless otherwise noted. All water used in biological experiments
was nanopure water obtained from Barnstead Nanopure Diamond
(Waltham, Mass.). Cell culture media, Dulbecco's modified Eagle's
medium ("DMEM") and fetal bovine serum ("FBS") were purchased from
Invitrogen (Carlsbad, Calif.).
[0132] Instruments.
[0133] All the denpols were characterized by nuclear magnetic
resonance ("NMR") and the molecular weight and molecular weight
distribution of denpol backbone was measured by gel permeation
chromatography ("GPC"). .sup.1H NMR spectra were recorded at 500
MHz on Bruker instruments. .sup.1H NMR chemical shifts were
reported as values in ppm relative to deuterated solvents D.sub.2O
(4.80). GPC was performed on an Agilent 1100 SEC system using a
OHpak SB-803 HQ column from Shodex, and the molecular weight was
determined with respect to poly(ethylene glycol) ("PEG") standards
purchased from Aldrich. DMF with 0.1% LiBr (wt/v) was used as the
eluent at a flow rate of 1.0 mL/min with column temperature at
45.degree. C. The size and zeta potential of denpol/siRNA
polyplexes were measured at 633 nm using Zetasizer (NanoZS) dynamic
light scattering instrument (Malvern Instruments, Malvern, UK) at
25.degree. C. with detection angle of 173.degree.. The nanoparticle
formed by denpol/siRNA complexes was visualized on a FEI/Philips
CM-20 conventional TEM operated at an accelerating voltage of 200
kV. The flow cytometry data was obtained on a Becton-Dickinson LSR
II flow cytometer with argon ion excitation laser at 488 nm
(Becton-Dickinson, Franklin Lakes, N.J.). Confocal fluorescence
images were acquired using a Zeiss LSM 510 inverted laser-scanning
confocal microscope.
[0134] Procedure for Denpol Functionalization:
[0135] In a one drum glass vial were added unfunctionalized denpol
(30 mg) at a desired generation, and two different boc-protected
amino acids at a specified ratio. DMF (1 mL) was added to dissolve
the solids, followed by adding BOP (1.05 equiv to the primary
amines) and DIPEA (1.05 equiv to the primary amines). The reaction
was left to stir for 24 hours at ambient temperature. Protected
denpol was precipitated in an excess amount of deionized water.
After removing water completely, the solid was dissolved in TFA (3
mL), DCM (1 mL) and triisopropylsilane (0.1 mL) as the scavenger.
After stirring overnight, excess TFA and DCM was removed in vacuo,
the resulting polymer was redissovled in methanol and precipitated
in ether. The precipitate was dissolved in water and lyophilized to
give a white powder. All denpols were characterized by .sup.1H NMR.
The functionalization ratio in NMR was calculated by comparing the
characteristic side chain peak with the aliphatic region in
lysine.
[0136] Gel Electrophoresis.
[0137] The binding of siRNA to denpol was studied by agarose gel
electrophoresis. Both siRNA and denpol were diluted with 10 mM
phosphate buffer (pH 7.4). Different amounts of a denpol solution
(5 mg/mL) were added to a 4 .mu.M siRNA solution (5.0 .mu.L) to
achieve different N/P ratios (the molar ratio of primary amine
groups from denpol and phosphate groups from siRNA, imidazole
groups of histidine residues were not counted because they are not
protonated at pH 7.4). The same buffer was added to adjust the
final volume to 10.0 .mu.L, followed by a 30 minute incubation at
room temperature. 6.times. gel loading dye (2.5 .mu.L) was added to
each sample and 10 .mu.L of the mixture was loaded into each well
of a 1% agarose gel with 1.times.GelRed.TM. dye. Electrophoresis
was run in TAE buffer (pH 7.9) at 60 V for 45 min and the gel was
visualized under a UV transilluminator.
[0138] Dextran Sulfate Competitive Binding Assay.
[0139] The binding strength of siRNA to denpol was studied by
competitive binding assay with dextran sulfate ("DS"). To a 4 .mu.M
siRNA solution (5 .mu.L) was added different denpol solutions at
N/P 40 and incubated for 1 hour at ambient temperature. A DS
solution (1 .mu.L) having different concentrations was added to the
complex to achieve the different S/P ratios (the molar ratio of
sulfate groups from DS and phosphate groups from siRNA) The mixture
was then incubated for another 30 minutes. The samples were then
subjected to agarose gel electrophoresis under the aforementioned
conditions.
[0140] Glutathione Triggered Release of siRNA from Denpol
Complexes.
[0141] To a 4 .mu.M siRNA solution (5 .mu.L) was added a
concentrated denpol solution to achieve N/P 40 and the final volume
was adjusted to 10 .mu.L by adding phosphate buffer (pH 7.4). After
a 1 hour incubation at room temperature, 55 mM glutathione (GSH) (1
.mu.L) was added to the polyplex solution to achieve a 5 mM final
concentration that was then followed by a 30 minute incubation at
ambient temperature. All samples were then subjected to agarose gel
electrophoresis under the aforementioned conditions.
[0142] DLS Measurements.
[0143] The size and zeta potential of denpol/siRNA polyplexes were
measured at 633 nm using a Zetasizer (NanoZS) dynamic light
scattering instrument (Malvern Instruments, Malvern, UK) at
25.degree. C. with detection angle of 173.degree.. Both denpol and
siRNA were diluted in nanopure water, and a denpol solution (50
.mu.L) was added to a 1.5 .mu.M siRNA solution (50 .mu.L; N/P 40),
followed by brief vortexing. After incubating at ambient
temperature for 30 minutes, a DLS measurement was taken. The
solution was then diluted with PBS (600 .mu.L), and subjected to
zeta-potential measurement. For PEGylated denpol studies, branched
PEI (M.sub.n=25k) was complexed with siRNA at N/P=15 and included
in the measurement as a control. At least three measurements were
taken for each sample and the mean values were reported.
[0144] TEM Characterization.
[0145] The nanoparticle formed by denpol/siRNA complexes was
visualized on a FEI/Philips CM-20 conventional TEM operated at an
accelerating voltage of 200 kV. Samples were prepared by placing a
drop of siRNA/denpol complex solution in DI water (1 mg/mL) on a
TEM grid (Ted Pella, Silicon Monoxide Type-A, 300 Mesh). The excess
solvent was removed by placing the sample on a filter paper. The
siRNA/denpol complexes were stained by placing a drop of 1.0 wt %
aqueous solution of uranyl acetate for 1 minute, followed by
removal of excess solvent.
[0146] Cell Culture.
[0147] NIH 3T3 fibroblast cells with or without green fluorescent
protein (GFP) were cultured in a standard CO.sub.2 incubator. The
culture media was DMEM with 10% fetal bovine serum (FBS). The cells
were trypsinized and passaged when they reached .about.90%
confluency.
[0148] MTT Assay.
[0149] NIH 3T3 fibroblast cells were seeded at a density of 5000
cells/well in 96-well plates for 24 hours. The culture media was
then changed from DMEM (100 .mu.L) with 10% fetal bovine serum
(FBS) to serum free DMEM (80 .mu.L) before the toxicity assay. A
PBS solution (20 .mu.L) containing different amounts of denpols
were added to each well, followed by a 24 hour incubation. The
media was then changed back to DMEM with 10% FBS and cultured for
another 48 hours. The media was replaced with a DMEM solution (50
.mu.L) containing 0.5 mg/mL MTT, followed by a 4 hour incubation at
37.degree. C. DMSO (100 .mu.L) was added to the solution to
dissolve the formed fomazan. A MTT reading was obtained by using a
plate reader (Abs 540 nm). As a positive control, cells were also
treated with poly(ethylene imine) ("PEI") at different
concentrations under the same conditions.
[0150] Transfection Screening.
[0151] NIH 3T3 fibroblast cells expressing GFP were seeded at a
density of 5000 cells/well in 96-well plates for 4 hours. Prior to
transfection, the media was replaced with serum free DMEM (80
.mu.L). Different polyplex solutions (20 .mu.L) at various N/P
ratios were added to each well to make the final siRNA
concentration of 100 nM, which was followed by incubating at
37.degree. C. for 4 hours. The media was changed back to DMEM with
10% FBS. After 48 hours, the fluorescence of each well was measured
by using a plate reader (Ex. 460 nm, EM. 525 nm). After the
fluorescence reading, cell viability was measured by a MTT assay
and the fluorescence was then normalized by percent viability to
eliminate toxicity-induced GFP reduction. As positive controls, two
bench-mark vectors, PEI and Lipofectamine.RTM. RNAiMAX were also
included in the study, and the transfection was carried out
following the manufacturer's protocol (100 nM siRNA
concentration).
[0152] Flow Cytometry Analysis of Transfected Cells.
[0153] NIH 3T3 fibroblast cells expressing GFP were seeded at a
density of 10,000 cells/well in 48-well plates for 24 hours. Prior
to transfection, the media was replaced with serum free DMEM (200
.mu.L). Different siRNA/denpol complex solutions (50 .mu.L) were
added to each well to make the final siRNA concentration 100 nM.
After incubating for 4 or 24 hours, the media was changed back to
DMEM with 10% serum and cultured for another 24 or 48 hours. Before
the analysis, cells were released from each well by trypsin and
harvested by centrifugation. The GFP fluorescence of transfected
cells was measured on a Becton-Dickinson LSR II flow cytometer with
argon ion excitation laser at 488 nm (Becton-Dickinson, Franklin
Lakes, N.J.). For each sample, data representing 10,000 objects
were collected as a list-mode file and analyzed using FACSDiva.TM.
software (Becton Dickinson, version 6.1.3). The transfection
efficiency was presented by comparing the fluorescence of
transfected cells with untreated cells.
[0154] Confocal Laser Scanning Microscopy.
[0155] Confocal laser scanning microscopy was used to track labeled
siRNA in the transfected cells. Unmodified NIH 3T3 fibroblast cells
were seeded at a density of 15000 cells/well on an 8-well chamber
slide (Lab-Tek, Rochester, N.Y.) for 24 hours. Cy3-labeled siRNA
was complexed with different denpols at N/P 80 and used to
transfect the cells under the aforementioned conditions. After
transfection, the media was switched back to DMEM with 10% serum.
Confocal fluorescence spectroscopy was performed at different time
points after the transfection. The nucleus was counter-stained with
DAPI. All confocal images were acquired using a Zeiss LSM 510
inverted laser-scanning confocal microscope. A 40.times.numerical
aperture of 1.4 oil immersion planapochromat objective was used for
all experiments. A 559 nm helium-neon laser, a SMD640 dichroic
mirror, and a 575-620 nm band-pass barrier filter were used to
obtain the images of Cy3-labeled siRNA. Images of DAPI-stained
nuclei were acquired using a 780 nm two-photon excitation light, a
635 nm dichroic mirror, and a 655-755 nm band-pass barrier filter.
The two fluorescent images were scanned separately and overlaid
together with the differential interference contrast image (DIC).
The cells were scanned as a z-stack of two-dimensional images
(1024.times.1024 pixels) and an image cutting approximately through
the middle of the cellular height was selected to present the
intracellular siRNA localization.
[0156] Statistical Analysis.
[0157] All quantitative assays were performed in triplicates, data
were expressed as mean.+-.SEM.
[0158] Small Focused Library Screening Assays.
[0159] To demonstrate the proof of concept, a small focused library
of amino acid based denpol by a "graft-from" approach as presented
in SCHEME 5:
##STR00021## ##STR00022##
[0160] The polymer backbone was synthesized by step-growth
polymerization of dicysteine 1 and L-lysine 2 using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide ("EDC") as the
coupling reagent (polymer M.sub.n.about.15 kD, PDI.about.1.8 by
GPC). Dicysteine was introduced into the polymer backbone as the
environmentally responsive motif for facilitated siRNA release in
the cytoplasm. Onto the linear polymer backbone 3, L-lysine-based
dendron was then grown generation by generation through
solution-phase peptide coupling. Finally, after reaching the
desired generation 4, the outer layer of the dendron was
functionalized by a combination of one hydrophilic and one
hydrophobic amino acid at various ratios 5. Using the combinatorial
synthesis method, three hydrophilic amino acids (lysine: K, serine:
S, and histidine: H) and four hydrophobic amino acids (tryptophan:
W, phenylalanine: F, tyrosine: Y, and leucine: L) were used to
quickly generate a small focused library of G1-G2 amphiphilic
denpols. For simplicity, the denpols were named after the
generation of the dendron and the compositions of the amphiphilic
amino acids on the outer layer using one-letter amino acid codes.
For example, G2 75H-25W represents a denpol carrying multivalent
second-generation denrons having 75 mol % histidine and 25 mol %
tryptophan incorporated on the outer layer.
[0161] Denpol Synthesis and Characterization.
[0162] Following SCHEME 6, polymer backbone 1, G1 Denpol 2 and G2
Denpol 3 were generated, which was further functionalized to make
functionalized G2 Denpol:
##STR00023## ##STR00024##
[0163] Synthesis of Polymer Backbone 1:
[0164] H-Lys-OEt.2HCl (2.471 g, 10.0 mmol), (Boc-Cys-OH).sub.2
(4.405 g, 10.0 mmol) were dissolved in DMSO (15.0 mL) in a wide,
shallow glass jar. Once the amino acids were dissolved after
vigorous stirring, EDC.HCl (11.502 g, 60.0 mmol), HOBt (2.973 g, 22
mmol), and DIPEA (4.355 mL, 25 mmol) were added to the reaction.
The reaction vessel was capped and the mixture was left to stir at
ambient temperature for 48 hours. One drop of the crude reaction
mixture was diluted with DMF and subjected to GPC analysis. Crude
polymer was precipitated by adding the reaction mixture to water
and then separated by centrifugation. After completely removing
water, the polymer was dissolved in trifluoroacetic acid ("TFA")
(50 mL) and DCM (10 mL) and left to stir for 24 hours at ambient
temperature. After deprotection, the solvent was removed with
rotary evaporation and the resulting solid was re-dissolved in
methanol. Deprotected polymer was then obtained by precipitation of
the methanol solution in diethyl ether. The polymer was further
purified by dialysis against methanol for 2 days with MWCO 3,500.
After removing methanol in vacuo, the polymer was lyophilized to
give a white powder. (3.30 g, 56% yield) .sup.1H NMR (500 MHz,
D.sub.2O): .delta. 4.52-4.34 (2H), 4.34-4.18 (.sup.3H), 3.47-3.12
(6H), 2.0-1.86 (1H), 1.86-1.68 (1H), 1.68-1.52 (2H), 1.52-1.37
(2H), 1.34-1.23 (t, 3H). The protected polymer was characterized by
GPC with 0.1% LiBr DMF as the eluent, poly(ethylene glycol) ("PEG")
standards were used as the reference: M.sub.n=15.0 kDa,
M.sub.w/M.sub.n=1.76.
[0165] Synthesis of G1 Denpol 2.
[0166] In a round bottom flask (25 Ml), 2.20 g of 1 (3.72 mmol, 1
equiv) and 3.93 g boc-lys(boc)-OH.DCHA (7.44 mmol, 2 equiv) were
dissolved in DMF (10 mL). After which, DIPEA (8.18 mmol, 2.2 equiv)
(1.42 mL) and BOP (8.18 mmol, 2.2 equiv) (3.62 g) were added. The
reaction was left to stir overnight at ambient temperature. The
protected denpol was then precipitated by pouring the solution into
water. After completely removing water, the polymer was dissolved
in a TFA/DCM solution (3:1) (20 mL) and stirred overnight at
ambient temperature. Excess TFA and DCM were removed in vacuo. The
resulting polymer was redissovled in methanol and precipitated in
ether. The precipitate was dissolved in water and lyophilized to
give a white powder. (3.29 g, 81% yield) .sup.1H NMR (500 MHz,
D.sub.2O): .delta. 4.74-4.55 (2H), 4.38-4.24 (1H), 4.24-4.13 (2H),
4.03 (2H), 3.28-2.88 (10H), 1.97-1.26 (18H), 1.23 (3H).
[0167] Synthesis of G2 Denpol 3.
[0168] In a round bottom flask (25 mL), 2.00 g of 2 (1.81 mmol, 1
equiv) and 3.81 g boc-lys(boc)-OH.DCHA (7.22 mmol, 4 equiv) were
dissolved in DMF (10 mL). After which, DIPEA (7.60 mmol, 4.2 equiv)
(1.30 mL) and BOP (7.60 mmol, 4.2 equiv) (3.36 g) were added. The
reaction was left to stir overnight at ambient temperature. The
protected denpol was then precipitated by pouring the solution into
water. After completely removing water, the polymer was dissolved
in a TFA/DCM solution (3:1) (20 mL) and stirred overnight at
ambient temperature. Excess TFA and DCM were removed in vacuo. The
resulting polymer was re-dissolved in Methanol and precipitated in
ether. The precipitate was dissolved in water and lyophilized to
give a white powder. (3.20 g, 85% yield). .sup.1H NMR (500 MHz,
D.sub.2O): .delta. 4.74-4.63 (.sup.1H), 4.63-4.49 (1H), 4.42-4.23
(3H), 4.23-4.10 (2H), 4.06-3.96 (2H), 3.90 (2H), 3.29-2.89 (18H),
1.98-1.26 (42H), 1.23 (3H).
[0169] G1 Denpol:
##STR00025##
[0170] G1 75K-25F: white powder, 92% yield, .sup.1H NMR (D.sub.2O):
.delta. 7.37 (3H), 7.24 (2H), 4.73-3.84 (11H), 3.33-2.84 (18H),
2.01-1.12 (39H). Percent functionalization by .sup.1H NMR: 75%
K-23% F. G1 75K-25W: white powder, 71% yield, .sup.1H NMR
(D.sub.2O): .delta. 7.84-6.93 (5H), 4.48-3.83 (11H), 3.50-2.66
(18H), 2.13-0.77 (39H). Percent functionalization by .sup.1H NMR:
75% K-19% W.
[0171] G1 75H-25F: white powder, 87% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.66 (3H), 7.67-6.92 (8H), 4.64-3.90 (11H), 3.49-2.77
(18H), 2.06-1.04 (21H). Percent functionalization by .sup.1H NMR:
68% H-26% F.
[0172] G1 75H-25W: white powder, 91% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.79-8.32 (3H), 7.73-6.84 (8H), 4.66-3.88 (11H), 3.46-2.87
(18H), 1.98-1.06 (21H). Percent functionalization by .sup.1H NMR:
75% H-25% W.
[0173] G1 75H-25Y: white powder, 87% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.64 (3H), 7.48-7.24 (3H), 7.17-6.93 (2H), 6.91-6.67 (2H),
4.67-3.96 (11H), 3.45-2.92 (18H), 1.95-1.06 (21H). Percent
functionalization by .sup.1H NMR: 70% H-24% Y.
[0174] G2 Denpol:
##STR00026##
[0175] G2 25K-75F: white powder, 67% yield, .sup.1H NMR (D.sub.2O):
.delta. 7.35 (18H), 7.24 (12H) 4.66-3.83 (19H), 3.33-2.80 (34H),
2.01-0.93 (57H). Percent functionalization by .sup.1H NMR: 25%
K-62% F.
[0176] G2 50K-50F: white powder, 63% yield, .sup.1H NMR (D.sub.2O):
.delta. 7.38 (12H), 7.27 (8H), 4.69-3.88 (19H), 3.40-2.86 (34H),
2.05-0.98 (69H). Percent functionalization by .sup.1H NMR: 50%
K-45% F.
[0177] G2 50H-50F: white powder, 79% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.48 (4H), 7.28-6.93 (24H), 4.67-3.77 (19H), 3.33-2.60
(34H), 1.86-0.76 (45H). Percent functionalization by .sup.1H NMR:
41% H-41% F.
[0178] G2 75K-25F: white powder, 84% yield, .sup.1H NMR (D.sub.2O):
.delta. 7.33 (6H), 7.23 (4H), 4.73-3.87 (19H), 3.36-2.80 (34H),
2.07-0.92 (81H). Percent functionalization by .sup.1H NMR: 75%
K-23% F.
[0179] G2 75K-25W: white powder, 92% yield, .sup.1H NMR (D.sub.2O):
.delta. 7.63-6.91 (10H), 4.62-3.79 (19H), 3.53-2.61 (34H),
2.09-0.68 (81H). Percent functionalization by .sup.1H NMR: 75%
K-23% W.
[0180] G2 75S-25F: white powder, 93% yield, .sup.1H NMR (D.sub.2O):
.delta. 7.34 (6H), 7.23 (4H), 4.73-4.02 (19H), 4.02-3.81 (12H),
3.31-2.85 (22H), 1.93-0.98 (45H). Percent functionalization by
.sup.1H NMR: 73% S-22% F.
[0181] G2 75H-25F: white powder, 69% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.50 (6H), 7.31 (16H), 4.87-3.99 (19H), 3.44-2.83 (34H),
2.00-1.11 (45H). Percent functionalization by .sup.1H NMR: 75%
H-25% F.
[0182] G2 75H-25W: white powder, 78% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.64 (6H), 7.74-6.78 (16H), 4.46-3.86 (19H), 3.47-2.63
(34H), 2.04-0.80 (45H). Percent functionalization by .sup.1H NMR:
75% H-21% W.
[0183] G2 75H-25Y: white powder, 76% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.66 (6H), 7.38 (6H), 7.05 (4H), 6.76 (4H), 4.63-3.95
(19H), 3.48-2.77 (34H), 1.97-1.05 (45H). Percent functionalization
by .sup.1H NMR: 75% H-20% Y.
[0184] G2 75H-25L: white powder, 79% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.68 (6H), 7.39 (6H), 4.48-3.84 (19H), 3.48-2.87 (30H),
1.99-1.10 (51H), 0.90 (12H). Percent functionalization by .sup.1H
NMR: 75% H-22% L.
[0185] G2 88H-12F: white powder, 69% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.77-8.25 (7H), 7.31 (10H), 7.20 (2H), 4.72-4.01 (19H),
3.42-2.83 (34H), 1.92-1.09 (45H). Percent functionalization by
.sup.1H NMR: 83% H-15% F.
[0186] G2 88H-12W: white powder, 84% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.72-8.26 (7H), 7.63-6.88 (12H), 4.74-3.96 (19H), 3.43-2.70
(34H), 1.94-1.06 (45H). Percent functionalization by .sup.1H NMR:
88% H-12% W.
[0187] G2 88H-12Y: white powder, 76% yield, .sup.1H NMR (D.sub.2O):
.delta. 8.80-8.50 (7H), 7.41 (7H), 7.10 (2H), 6.95-6.72 (2H),
4.66-4.00 (19H), 3.51-2.82 (34H), 1.89-1.18 (45H). Percent
functionalization by .sup.1H NMR: 86% H-11% Y.
[0188] PEGylated Denpol Synthesis:
[0189] By slightly modifying the denpol synthesis procedure above,
PEGylated denpols were generated. 5% PEG denpol can be synthesized
by following the reactions presented in SCHEME 7. Moreover,
additional percentages of PEG (e.g., 10% or 20%) were synthesized
by using SCHEME 4 but with a different molar ratio of PEG5000 for
incorporation.
##STR00027##
[0190] Synthesis of NHS-PEG5k 4:
[0191] Under nitrogen, 17.010 g (3.00 mmol, 1 equiv) of
poly(ethylene glycol) methyl ether (PEG5k, M.sub.n=5000,
Sigma-Aldrich) and triethylamine (TEA) (0.460 mL, 3.3 mmol, 1.1
equiv) were dissolved in 30 mL anhydrous DCM in a 100 mL round
bottom flask. The solution was stirred for 30 min on ice. Under
nitrogen, N,N'-disuccinimidyl carbonate (1.153 g, 4.50 mmol, 1.5
equiv) was suspended in 5 mL anhydrous DCM in another flask. Using
an ice bath, the PEG5k/TEA solution was added dropwise to the flask
containing N,N'-Disuccinimidyl carbonate under nitrogen. The
reaction was left to stir for 30 min on ice and another 24 hours at
room temperature. After the reaction, the solution was diluted with
chloroform and washed with ice cold brine (3.times.). The organic
layer was dried over sodium sulfate and all solvents were removed
in vacuo to afford the NHS-PEG5K as a white solid (10.2 g, 60%).
.sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 4.50 (m, 2H), 4.38-4.52
(m, 496H), 3.42 (s, 3H), 2.88 (s, 4H).
[0192] Synthesis of Polymer Backbone 1:
[0193] H-Lys-OEt.2HCl (2.471 g, 10.0 mmol), (Boc-Cys-OH).sub.2
(4.405 g, 10.0 mmol) were dissolved in DMSO (15.0 mL) in a wide,
shallow glass jar. Once the amino acids were dissolved after
vigorous stirring, EDC.HCl (11.502 g, 60.0 mmol), HOBt (2.973 g, 22
mmol), and DIPEA (4.355 mL, 25 mmol) were added to the reaction
mixture. The reaction vessel was capped and the mixture was left to
stir at room temperature for 48 hours. One drop of the crude
reaction mixture was diluted with DMF and subjected to GPC
analysis. Crude polymer was precipitated by adding the reaction
mixture to water and then separated by centrifugation. After the
water was completely removed, the polymer was dissolved in 50 mL
trifluoroacetic acid (TFA) and 10 mL DCM and left to stir for 24
hours at room temperature. After deprotection, the solvent was
removed with rotary evaporation and the resulting solid was
re-dissolved in methanol. Deprotected polymer was then obtained by
precipitating the methanol solution in diethyl ether. The polymer
was further purified by dialysis against methanol for 2 days with
MWCO 3,500. After removing the methanol in vacuo, the polymer was
dissolved in water and lyophilized to give a white powder. (3.30 g,
56% yield) .sup.1H NMR (500 MHz, D.sub.2O): .delta. 4.52-4.34 (2H),
4.34-4.18 (3H), 3.47-3.12 (6H), 2.0-1.86 (1H), 1.86-1.68 (1H),
1.68-1.52 (2H), 1.52-1.37 (2H), 1.34-1.23 (t, 3H). The protected
polymer was characterized by GPC with 0.1% LiBr DMF as the eluent,
poly(ethylene glycol) (PEG) standards were used as the reference:
M.sub.n=15.0 kDa, M.sub.w/M.sub.n=1.76.
[0194] Synthesis of 5% PEG-G1-NH.sub.2 Denpol 5.
[0195] Under nitrogen and in an oven-dried 10 mL round bottom
flask, polymer backbone 1 (100 mg, 0.160 mmol repeating units, 1.00
equiv) and NHS-PEG5k 4 (80 mg, 0.016 mmol, 0.10 equiv) were
dissolved in 2 mL anhydrous DMF followed by addition of 14 .mu.L
DIPEA (0.080 mmol, 0.5 equiv). The reaction was left to stir under
nitrogen overnight. Without purification, the reaction was diluted
with 2.0 mL DMF, followed by the addition of Boc-lys(boc)-OH.DCHA
(168 mg, 0.320 mmol, 2.00 equiv), BOP (142 mg, 0.320 mmol, 2.00
equiv) and DIPEA (56 .mu.L, 0.320 mmol, 2.00 equiv). The reaction
was stirred at room temperature for another 24 hours. Afterwards,
the solution was diluted with methanol and dialyzed against
methanol for 2 days with MWCO 8000 membrane. After removing the
methanol in vacuo, the protected polymer was dissolved in a 5 mL
TFA/DCM solution (5:1) and stirred overnight at room temperature.
Excess TFA and DCM was removed in vacuo, the resulting polymer was
re-dissolved in methanol and dialyzed against methanol again with
MWCO 8000 membrane. After removing the methanol, the deprotected
polymer was dissolved in water and lyophilized to give a white
powder. (162 mg, 63% yield).sup.1H NMR (500 MHz, D.sub.2O): .delta.
4.40 (1H), 4.24 (2H), 4.11 (2H), 3.80-3.60 (77H, PEG,
--OCH.sub.2CH.sub.2O--, 7 mol %), 3.42 (0.42H, PEG, --OMe, 7 mol
%), 3.31-3.00 (8H), 2.05-1.33 (16.48H, lys-dendron, 92 mol %), 1.28
(3H).
[0196] Synthesis of 5% PEG-G2-NH.sub.2 denpol 6.
[0197] In a 5 mL round bottom flask, 5% PEG-G1-NH2 5 (150 mg, 0.094
mmol, 1.00 equiv) and boc-lys(boc)-OH.DCHA (198.4 mg, 0.376 mmol,
4.00 equiv) was dissolved in 5 mL DMF, followed by the addition of
DIPEA (69 .mu.L, 0.395 mmol, 4.20 equiv) and BOP (174.7 mg, 0.395
mmol, 4.2 equiv). The reaction was left to stir overnight at room
temperature. Afterwards, the solution was diluted with methanol and
dialyzed against methanol for 2 days with MWCO 8000 membrane. After
removing the methanol in vacuo, the protected polymer was dissolved
in a 5 mL TFA/DCM solution (5:1) and stirred at overnight at room
temperature. Excess TFA and DCM was removed in vacuo, the resulting
polymer was redissovled in methanol and dialyzed against methanol
again with MWCO 8000 membrane. After removing MeOH, the deprotected
polymer was dissolved in water and lyophilized to give a white
powder. (195 mg, 73% yield). .sup.1H NMR (500 MHz, D.sub.2O):
.delta. 4.60 (1H), 4.40-4.20 (5H), 4.01 (1.82H), 3.89 (1.89H),
3.83-3.50 (77.8H, PEG, --OCH.sub.2CH.sub.2O--), 3.38 (0.46H, PEG,
--OMe), 3.31-3.05 (7.4H), 2.98 (9.15H), 2.05-1.33 (39.8H,
lys-dendron), 1.25 (3H).
[0198] Synthesis of 5% PEG-G2-75H25W 7:
[0199] In a one drum glass vial, 5% PEG-G2-NH.sub.2 6 (30 mg,
0.0105 mmol, 1.00 equiv), Boc-His(boc)-OH.DCHA (33.8 mg, 0.0630
mmol, 6.00 equiv) and Boc-Trp(boc)-OH (8.5 mg, 0.0210 mmol, 2.00
equiv) were dissolved in DMF (1.5 mL), followed by the addition of
DIPEA (15 .mu.L, 0.0861 mmol, 8.20 equiv) and BOP (38.1 mg, 0.0861
mmol, 8.20 equiv). The reaction was left to stir at rt for 24
hours. Protected denpol was purified by dialysis against methanol
with 8000 MWCO membrane. After removing the methanol, the solid was
dissolved in TFA (3 mL), DCM (1 mL) and triisopropylsilane (0.1 mL)
as the scavenger. After stirring overnight, excess TFA and DCM were
removed in vacuo, the resulting polymer was re-dissolved in
methanol and dialyzed against methanol with a MWCO 8000 membrane.
After removing the methanol, the deprotected polymer was dissolved
in water and lyophilized to give a white powder. (40.7 mg, 88%
yield). .sup.1H NMR (500 MHz, D.sub.2O): .delta. 8.05-7.70 (5H,
His), 7.60-6.80 (13.8H, His+Trp), 4.60 (1.2H), 4.40-4.00 (16H),
3.83-3.50 (71.3H, PEG, --OCH.sub.2CH.sub.2O--), 3.38 (0.68H, PEG,
--OMe), 3.31-2.80 (31.6H), 1.95-0.90 (45H, lys-dendron).
[0200] PEGylation Ratio and Functionalization Ratio of Different
Denpols:
[0201] The ratio of PEG incorporation was calculated by comparing
the methyl peak (--OCH.sub.3) and methylene peaks
(--OCH.sub.2CH.sub.2O--) of the PEG with ethyl ester peak
(--COOCH.sub.2CH.sub.3) of the polymer backbone in G1 denpol (see
FIG. 6A). The functionalization ratio of histidine and tryptophan
was calculated by comparing the characteristic side chain peak with
the aliphatic region in lysine (see FIG. 6B). TABLE 1 summarizes
the characterization of the three different PEG denpols.
TABLE-US-00001 TABLE 1 Functionalization ratio of different
PEGylated denpols Sample PEG 5k Dendron His Trp 5% PEG-G2-75H25W 7%
92% 68% 24% 10% PEG-G2-75H25W 12% 86% 75% 25% 20% PEG-G2-75H25W 19%
80% 69% 25% *All ratios were molar ratio based on .sup.1H NMR
analysis.
[0202] Assessing Denpols/siRNA Complexes.
[0203] The binding capability of the denpols with siRNA was
initially assayed by using gel electrophoresis, and most denpols
could completely complex with siRNA at an N/P ratio from
10.about.30. The binding strength was further evaluated by
competitive binding assays using an anionic polymer, dextran
sulfate (DS, MW=25 kD) as the challenger (selected examples are
shown in FIG. 7A-C). For this purpose, polyplexes prepared at N/P
40 were incubated with different amount of DS to compete with
siRNA. A number of trends were observed in the competitive binding
assay: (1) the generation of dendron is important for binding
strength to siRNA; (2) the second generation denpol has much
stronger binding strength than first generation (see FIG. 7A-B),
due to the increased multivalent binding sites from the dendrons;
and (3) the composition of amino acids on the outer layer also has
a significant impact on the binding capability to siRNA. In our
small library of denpols, tryptophan (W) incorporated denpols show
the strongest binding to siRNA. For example, no appreciable siRNA
release could be observed for G2 75H-25W at S/P up to 30 (FIG. 7C).
As previously reported, the indole ring on tryptophan could
intercalate into nucleotide base pairs (e.g., see Rajeswari et al.,
Biochemistry 1987, 26, 6825; and Rajeswari et al., Biochemistry
1992, 31, 6237). These interactions could account for the increased
binding affinity of tryptophan functionalized denpol to siRNA.
Lastly, due to the dicysteine building blocks on the polymer
backbone, complexed siRNAs can be released from the polyplexes by
the addition of a reducing agent. For this assay, different
denpol/siRNA polyplexes were treated with glutathione (GSH) at a
concentration close to physiological conditions (5 mM). Gel assays
demonstrate that siRNA was completely released from most denpols
after the GSH treatment (see FIG. 7D). For the tryptophan
containing G2 denpol, which has the strongest binding affinity to
siRNA, the binding strength was significantly reduced. Presumably,
the reduced binding affinity to siRNA after GSH treatment is due to
the reduction-triggered degradation of the denpol, which decreases
the multivalency for binding. GSH triggered release or exposure to
a reducing environment in the cell should be beneficial for
intracellular siRNA delivery, by releasing the siRNA from denpol
once inside the cell. Both transmission electron microscope ("TEM")
and atomic force microscope ("AFM") images show denpol/siRNA
polyplexes as spherical nanoparticles with 30-80 nm in diameter
(see FIG. 7E-F). Based on dynamic light scattering ("DLS"), most G2
denpols were able to condense with siRNA into nanoparticles with
diameter smaller than 100 nm in buffer solution (see FIG. 7G).
[0204] Studies to Determine the Size of Denpol/siRNA Polyplexes and
PEGylated Denpol/siRNA Polyplexes.
[0205] The size of the polyplexes was investigated by DLS and TEM.
The DLS results showed that most denpols were able to condense
siRNA into particles smaller than 100 nm in diameter (see FIG. 8
and FIG. 9). Further, the polyplexes have a moderate positive
charge (zeta-potential .about.15 mV). The PEGylated denpols form
smaller particles with siRNA, but the polydispersity also increases
(see FIG. 10). Also the PEGylation greatly reduces the surface
charge of the nanoparticle. With 10% PEGylated denpol, the surface
charge is near neutral, which therefore increases serum stability
of polyplexes and is very beneficial for systemic in vivo delivery.
FIG. 12C shows a representative DLS curve of polyplexes prepared
from G2 75H-25W at N/P 40 (z-avg=80 nm, PDI=0.317). TEM provides
direct visualization of the polyplexes. TEM shows the polyplexes as
spherical nanoparticles with diameters .about.20-80 nm (see FIG.
11, and FIG. 12A). After GSH treatment, however, no discreet
nanoparticle could be observed under TEM (see FIG. 12B), which
confirmed the particle responsiveness to reducing agent as observed
by gel assay (see FIG. 12D).
[0206] siRNA Transfection Studies in NIH 3T3 Cells Using
Denpols.
[0207] The efficacy of siRNA transfection was first screened using
an engineered NIH 3T3 cell line expressing enhanced green
fluorescence protein ("GFP"). siRNA against GFP was complexed with
different denpols at N/P 20-120 and used to transfect 3T3 cells
cultured in a 96-well plates. After incubating for 48 hours, the
GFP fluorescence of each well was measured by using a plate reader
and the cell viability was determined by an MTT assay. GFP
fluorescence was then normalized by percent viability to eliminate
toxicity-related GFP reduction. Two benchmark transfection agents,
branched poly(ethylene imine) ("PEI," MW.about.25 kD) and
Lipofectamine.RTM., were used as the positive controls. The
screening results of transfection efficiency for G2 denpols at
optimal N/P ratio are summarized in FIG. 14 (complete screening
data for G1 denpols and G2 denpols are shown in FIG. 15 and FIG.
16, respectively).
[0208] Denpols carrying both histidine and aromatic amino acids
show very high transfection efficiency and low cytotoxicity
(75H-25F, 88H-12W, 75H-25Y). Without aromatic groups (75H-25L), no
transfection could be observed. And without histidine, substantial
silencing only occurred at a very high ratio of hydrophobic amino
acid (25K-75F), which caused significant cytotoxicity.
[0209] On the basis of the initial screening results, several
denpols were selected for more detailed investigation. The
dose-dependent toxicity was first determined by MTT assay. As shown
in FIG. 12D, all the denpols are two orders of magnitude less toxic
than PEI, suggesting the denpols of the disclosure are a very safe
platform for siRNA delivery. PEGylation further reduces the
toxicity of the denpol system. With 5% PEGylation, the toxicity was
greatly reduced at high concentration, and with 10% and 20%
PEGylation, the polymers were non-toxic up to 2.5 mg/mL (see FIG.
17).
[0210] Flow Cytometry Analysis of siRNA Transfected NIH 3T3 Cells
Using Denpols.
[0211] The transfection of selected denpols was then repeated and
analyzed by flow cytometry for a more accurate measurement. The
results agreed well with the initial screening result and confirmed
that both histidine and aromatic groups were important for GFP
silencing (see FIG. 18A). Denpols with either K-F (lacking H) or
H-L (lacking aromatic residue) combinations didn't show any
significant gene knockdown. Scrambled siRNA/G2 75H-25W was also
transfected and showed minimal effect to GFP expression, indicating
high specificity and low off-target effect (see FIG. 18A).
[0212] siRNA Transfection Studies in NIH 3T3 Cells Using Denpols in
Serum Containing RNase.
[0213] For a successful in vivo delivery system, the polyplexes
must be able to protect siRNA from the RNase in the serum, and
should also have minimum aggregation with negatively charged
proteins in serum. In order to study the serum compatibility of the
current system, transfection of denpol/siRNA was carried out in
DMEM solution containing 10% to 75% fetal bovine serum. Denpol G2
75H-25W was chosen because it showed the highest siRNA binding
affinity and high transfection efficiency in serum-free condition.
At all serum concentrations tested, this denpol showed
significantly higher transfection efficiency over
Lipofectamine.RTM. (see FIG. 18B). Even at 75% serum concentration,
higher than 50% knockdown could still be observed for this
denpol.
[0214] In order to study the serum stability of the PEGylated
denpol complexes, in vitro transfection was carried out in DMEM
containing different concentration of fetal bovine serum. As shown
in FIG. 20, transfection efficiency of Lipofectamine.RTM. greatly
reduces as the serum concentration increases. And without
PEGylation, the denpol works well up to 50% serum concentration,
but efficiency drops significantly at higher serum concentration.
Encouragingly, with 5% or 10% PEGylated denpols, the serum showed
minimum effect on the transfection efficiency and high protein
knockdown (80%) could still be achieved at 100% concentration. The
low toxicity and high transfection efficiency in serum make the
PEGylated denpol ideal candidate for in vivo applications.
[0215] Intracellular Confocal Fluorescence Trafficking Studies
Using Cy3-Labelled siRNA and Select Denpols.
[0216] In order to gain insights into the role of different
functional groups, intracellular fluorescence trafficking study
using a Cy3-labeled siRNA. Three amphiphilic analogues of G2
denpols were chosen for comparative studies: one having both H and
an aromatic residue (G2 75H-25F), one having an aromatic residue
but no H (G2 75K-25F), and the last one having H but no aromatic
residue (G2 75H-25L). Various Cy3-labeled polyplexes were exposed
to 3T3 cells for 4 hours in serum free media and then replaced with
normal media with 10% serum. Confocal fluorescence images were
taken at different time points after the transfection. As shown in
FIG. 21 and FIG. 22, the aromatic residue (F) is important for
cellular uptake. While no siRNA internalization was observed with
H-L functionalized denpol (G2 75H-25L), both H-F and K-F
combinations show very effective cell uptake (G2 75H-25F, G2
75K-25F). On the other hand, the buffering capacity of histidine
was also important for successful delivery. In G2 75K-25F
transfected cells, the siRNA fluorescence greatly diminished after
6 hours, and no siRNA could be observed 24 hours after
transfection. In contrast, siRNA remained present in G2 75H-25F
transfected cells for up to 24 hours. It is hypothesized that the
buffering capacity of histidine could aid endosomal membrane
disruption through either "proton sponge" mechanism or increased
amphiphilicity. Without the pH responsive groups (G2 75K-25F),
endocytosed siRNA would likely be transported to lysosome, followed
by enzymatic degradation and fast clearance.
[0217] On the basis of the transfection and fluorescence
trafficking results, amphiphilicity of aromatic amino acids and the
buffering capacity of histidine could work synergistically for
effective siRNA delivery in a denpol system disclosed herein.
Denpols with aromatic amino acids (F, W, Y) showed effective
cellular uptake and transfection while no cellular uptake or
silencing was observed with leucine functionalized denpol (G2
75H-25L). Presumably, the relatively large aromatic hydrophobic
groups enhance cellular membrane interaction for the denpol
complexes.
[0218] Efficient endosomal escape is also important for successful
siRNA delivery as most vectors are internalized by endocytosis. The
buffering capacity of histidine aids endosomal membrane rupture
through either "proton sponge" mechanism or increased
amphiphilicity. Therefore, denpols comprising histidine can utilize
a low ratio of aromatic groups for effective delivery, while
denpols without histidine need a high ratio of incorporated
aromatic amino acids. The pH responsiveness ensures the
biocompatibility of denpols at neutral pH and also increases
membrane lysis at an acidic pH (i.e., endosome) to facilitate
endosomal escape.
[0219] A number of embodiments have been described herein.
Nevertheless, it will be understood that various modifications may
be made without departing from the spirit and scope of this
disclosure. Accordingly, other embodiments are within the scope of
the following claims.
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